WO2021078934A1 - (be-)curtovirus replicon-mediated genome editing in plants - Google Patents

(be-)curtovirus replicon-mediated genome editing in plants Download PDF

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WO2021078934A1
WO2021078934A1 PCT/EP2020/079874 EP2020079874W WO2021078934A1 WO 2021078934 A1 WO2021078934 A1 WO 2021078934A1 EP 2020079874 W EP2020079874 W EP 2020079874W WO 2021078934 A1 WO2021078934 A1 WO 2021078934A1
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sequence
plant
cell
genome
genome editing
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Nadine SCHUMANN
Markus Niessen
Omid Eini
Mark VARRELMANN
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KWS SAAT SE & Co. KGaA
Institut Für Zuckerrübenforschung Des Vereins Der Zuckerindustrie E.V.
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
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Definitions

  • the present invention relates to the technical field of targeted modification of a nucleotide sequence of interest in the genome of a plant. More particularly, the invention relates to the provision of new viral replicons for delivering genome editing tools to plant cells recalcitrant for transformation for, in particular, achieving homology-directed repair (HDR)-mediated genome editing suitable for, inter alia, plant cells or plants of the species of the family Amaranthacea, preferably a cell of a plant from the genus Beta or Spinacia, and/or species of the family Solanacea, particularly from the genus Nicotiana.
  • HDR homology-directed repair
  • the invention thus describes a method for increased efficiency of genome editing, in particular HDR-mediated genome editing, in a plant or a plant cell by providing specific viral replicons facilitating the modification of transformation recalcitrant plants and plant cells.
  • CRISPR-based genome editing While for many crops the use of CRISPR-based genome editing (GE) is described already, there are several crop plants for which only little is known about the successful and efficient methods for such genome editing, in particular complex modifications like HDR-mediated genome editing.
  • a plant of the species Beta vulgaris (such as, for example, sugar beet, fodder beet, red beet, or chard) is well known as being very recalcitrant in transgenic modifications.
  • Beta vulgaris such as, for example, sugar beet, fodder beet, red beet, or chard
  • There are only very few established methods for transformation mainly based on Agrobacterium- mediated transformation, which were established for recalcitrant plants, however, with a high level of genotype dependency.
  • the common way to modify a nucleotide sequence of interest in the genome of e.g. sugar beet is to stably transform plants via Agrobacterium- mediated delivery of a T-DNA vector expressing a nucleotide sequence of interest, which may be a gene conferring an important trait or a gene silencing cassette.
  • a T-DNA vector expressing a nucleotide sequence of interest which may be a gene conferring an important trait or a gene silencing cassette.
  • stable transformation and subsequent regeneration of transgenic plants is amongst the aforementioned technical hurdles extremely labor-intensive, time-consuming and cost-intensive.
  • the Agrobacterium- mediated construct insertion into the plant genome does not occur in a targeted way, but randomly which is not desired for obtaining a predictable GE outcome. Therefore, positional effects appear frequently.
  • HDR-mediated genome editing allows targeted insertion of larger nucleotide molecules, e.g. entire genes, regulatory elements or the replacement of one allele for another which for example carries one or more individual amino acid substitution.
  • Lozano-Duran (2016) Geminiviruses for biotechnology: the art of parasite taming. New Phytologist, 210(1), 58-64.
  • Another pleiotropic effect of constitutive replicon expression might be an increase in off-target modifications, generally not desired for precise GE in plants.
  • EP 2 861 737 B1 disclosed a method to increase the efficiency of GE events in plants, which are based on homology-directed repair.
  • the only virus used is the Mastrevirus bean yellow dwarf virus (BeYDV) and, again, only plant species which are known to be susceptible for genetic modification and simple to recover, namely Arabidopsis thaliana and Nicotiana tabacum, are used. Therefore, the approach disclosed is not suitable for efficiently transforming and modifying recalcitrant dicotyledonous plants, as already transformation with the disclosed viruses may be associated with problems due to incompatibility of the relevant host to be transformed and later the host genome to be modified.
  • BeYDV Mastrevirus bean yellow dwarf virus
  • Beta vulgaris plant is not a natural host for the Mastrevirus BeYDV, in sugar beet BeYDV- based GVRs cannot replicate and interact with host factors as efficient as in the model plants Nicotiana tabacum or Arabidopsis.
  • family of Geminiviridae is highly diverse in structure and function which otherwise necessarily entails that findings for one specific virus from a genus or species attributed to the Geminiviridae family cannot easily be applied to another genus due to large differences in genome organization and pathology.
  • BeYDV Bean Yellow Dwarf Virus
  • the genome of this Geminivirus encodes four genes (Rep, RepA, V1 and V2) and contains a long internal region (LIR) and a short internal region (SIR).
  • Rep, RepA, V1 and V2 The genome of this Geminivirus encodes four genes (Rep, RepA, V1 and V2) and contains a long internal region (LIR) and a short internal region (SIR).
  • LIR long internal region
  • SIR short internal region
  • BCTIV Beet curly top iran virus
  • This virus encodes five genes (V1 , V2 and V3 on the virion-sense strand and Rep and C2 genes on the complementary-sense strand).
  • V1 , V2 and V3 on the virion-sense strand and Rep and C2 genes on the complementary-sense strand encodes five genes (V1 , V2 and V3 on the virion-sense strand and Rep and C2 genes on the complementary-sense strand).
  • the genome sequence and structure of the virion-sense stand of Becurtoviruses is similar to Curtoviruses while the complementary-sense strand has similarity to the Mastreviruses.
  • BSCTV Beet severe curly top virus
  • BCTV Beet curly top virus
  • Curtoviruses are monopartite Geminiviruses that are vectored by leafhoppers, often in a species-specific manner and rather different to Mastreviruses.
  • BCTV encodes usually 7 genes (V1 , V2, V3 on the virion-sense strand and Rep, C2, C3 and C4 on the complementary- sense strand) without SIR sequence.
  • the present invention thus provides, in a first aspect, a method for the targeted modification of at least one genomic target sequence in a plant cell preferably from a plant of the family Amaranthaceae or Solanaceae, preferably a cell of a plant from the genus Beta or Spinacia or Nicotiana, more preferably a cell of a plant of the species Beta vulgaris, Spinacia oleracea, Nicotiana benthamiana or Nicotiana tabacum, wherein the method comprises the following steps: (a) introducing into the cell (i) at least one viral replicon, or a sequence encoding the same, the replicon being preferably constructed based on a Becurtovirus, or a Curtovirus genome, wherein the at least one viral replicon further comprises: (ii) at least one genome editing system comprising at least one nucleic acid guided or site-specific nuclease, nickase or an inactivated nuclease, or a sequence en
  • a plant, plant cell, tissue, organ, or seed obtainable by or obtained by a method according to the first aspect is provided.
  • a viral replicon for plant transformation wherein the viral replicon is encoded by a sequence according to SEQ ID NO: 4 or 6, or a sequence having at least sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
  • an expression construct comprising or encoding: (i) at least one viral replicon as defined in the above aspect and (ii) a vector, preferably a binary vector, more preferably a binary vector as defined in the above aspect, and (iii) at least one component of at least one genome editing system, wherein the genome editing system comprises or encodes: (a) at least one nucleic acid guided or site-specific nuclease, nickase or an inactivated nuclease, or at least one site-directed nuclease, nickase or an inactivated nuclease, and optionally at least one nuclear or organellar localization sequence; and optionally (b) at least one guide molecule for guiding the at least one nucleic acid guided nuclease, nickase or an inactivated nuclease and optionally for activating at least one reverse transcriptase; and (c) optionally at least one repair template; and (d) optionally at least one repair template; and (d
  • a use of a viral replicon optimized in tropism for a plant or plant cell of interest, or a use of an expression construct according to the above aspect is provided, for stimulating homology-directed repair by reverting a host plant cell into the S-phase of the cell cycle, and/or for increasing homology-directed repair-based genome editing.
  • Figure 1 shows viral DNA levels of full-length clones (A) and replicons (B).
  • Figure 1 A shows virus accumulation in sugar beet (SB) cotyledons six days post inoculation. Cotyledons of sugar beet seedlings were syringe-infiltrated with full-length clones of different SB- infecting Geminiviruses (BSCTV, BCTV, BMCTV, BCTIV). DNA levels were determined via qPCR six days post infiltration (dpi). Four biological replicates were tested per construct. Error bars represent standard deviations.
  • Figure 1 B shows replication intensities of GVRs in sugar beet.
  • Cotyledons of sugar beet seedlings were syringe-infiltrated with GVRs derived from BSCTV (BSCTVReplM), BCTIV (BCTIVReplM) or BeYDV (LSL). DNA levels were determined via qPCR ten days post infiltration using virus specific primers. A reference gene from Beta vulgaris was used for normalization. Three biological replicates were tested per replicon. Error bars represent standard deviations.
  • Figure 2 shows a schematic view of the dexamethasone (DEX)-inducible promoter system.
  • Figure 3 shows a schematic view of the binary vector pZFNnptll containing the BCTIV-based replicon (BCTIV-Rep/MCS in pZFNnptll).
  • Figure 4 shows a schematic view of the binary vector pZFNnptll containing the BSCTV-based replicon (BSCTV-Rep/MCS in pZFNnptll).
  • Figure 5 shows the design of the binary vectors used for in planta proof of LbCpfl mediated genome editing.
  • the LbCpfl _tDTcrRNA_ALS#1 (SEQ ID NO: 25) construct harbors expression cassettes for nptll, LbCpfl and crRNA_ALS#1 , which is directed against the target region BvALS#1 .
  • the crRNA is embedded in a Pol ll-driven RNA transcript as described by Zhong et al tension 2017 (doi: 10.1038/nchembio.2410).
  • the LbCpfl _RibocrRNA_ALS#1 (SEQ ID NO: 26) construct harbors expression cassettes for nptll, LbCpfl and a crRNA_ALS#1 , which is directed against the target region BvALS#1.
  • the crRNA is produced from a Pol II promoter using the ribozyme-based strategy.
  • FIG. 6 shows GFP transcript levels in sugar beet.
  • BCTIV-GVR and BSCTV-GVRs outperform BeYDV-GVR in terms of heterologous gene expression.
  • Cotyledons of sugar beet seedlings were syringe-infiltrated with GVRs derived from BeYDV (LSLR), BCTIV (BCTIVReplM) or BSCTV (BSCTVReplM), which contain a 35S::GFP expression cassette.
  • 35S::GFP expressed via a binary vector (pZFN-GFP) was used as non-GVR control.
  • Transcript levels of GFP were measured via qRT-PCR four days (A) or six days (B) post infiltration.
  • GFP expression was normalized against a reference gene from sugar beet. Three biological repeats were tested. Error bars represent standard deviations.
  • FIG. 7 shows GFP transcript levels in Nicotiana benthamiana. Nicotiana benthamiana leaves were syringe-infiltrated with GVRs derived from BeYDV (LSLR), BCTIV (BCTIVReplM) or BSCTV (BSCTVReplM), which contain a 35S::GFP expression cassette. 35S::GFP expressed via a binary vector (pZFN-GFP) was used as non-GVR control. Transcript level of GFP were measured via qRT-PCR six days post infiltration. GFP expression was normalized against a reference gene from N. benthamiana. Three biological replicates were tested. Error bar represent standard deviations.
  • Figure 8 shows that transcription of LbCpfl is increased in BCTIV-GVRs compared to the corresponding binary vector controls.
  • Figure 8/1 shows relative transcription levels for the LbCpf1_tDTcrRNA construct.
  • the samples with the numbers 21 , 22, 23, 24, 41 , 42, 43, 44, 101 , 102, 103, 104, 81 , 82, 83, 84, 141 , 142, 143 and 144 represent relative transcription levels zero days post infiltration (dpi).
  • the samples with the numbers 25, 26, 27, 28, 45, 46, 47, 48, 105, 106, 107, 108, 85, 86, 87, 88, 145, 146, 147, 148 represent relative transcription levels 6 dpi.
  • the samples 29, 30, 31 , 32, 49, 50, 51 , 52, 109, 110, 111 , 112, 89, 90, 91 , 92, 149, 150, 151 , and 152 represent relative transcription levels 12 dpi.
  • Sample 108 (boxed) was used as a reference (set as 1).
  • Figure 8/2 shows relative transcription levels for the LbCpfl _Ribo_crRNA construct.
  • the samples with the numbers 21 , 22, 23, 24, 41 , 42, 43, 44, 101 , 102, 103, 104, 61 , 62, 63, 64, 121 , 122, 123 and 124 represent relative transcription levels 0 dpi.
  • the samples with the numbers 25, 26, 27, 28, 45, 46, 47, 48, 105, 106, 107, 108, 65, 66, 67, 68, 125, 126, 127, 128 represent relative transcription levels 6 dpi.
  • the samples 29, 30, 31 , 32, 49, 50, 51 , 52, 109, 110, 111 , 112, 69, 70, 71 , 72, 129, 130, 131 , and 132 represent relative transcription levels 12 dpi.
  • Sample 108 (boxed) was used as a reference (set as 1).
  • Figure 9 shows transcription of LbCpfl is increased in plant tissue infiltrated with BSCTV-GVRs compared to the corresponding binary vector controls.
  • Cotyledons of sugar beet seedlings were syringe-infiltrated with GVRs derived from BSCTV (pZFNnptll-BSCTV-Repl), which contain genome editing components (LbCpfl or LbCpfl +crRNA).
  • GE components expressed via a binary vector (pZFNnptll) were used as non-GVR controls.
  • Figure 10 shows the repair template for BvALS.
  • Figure 11 shows increased DNA levels (from the LbCpfl -encoding gene) in BCTIV-GVRs infiltrated plants compared to the corresponding binary vector controls at 6 dpi and 12 dpi, respectively.
  • Cotyledons of sugar beet seedlings were syringe-infiltrated with GVRs derived from BCTIV (pZFNnptll-BCTIV-Repl), which contain genome editing components (LbCpfl , LbCpfl +tDT-crRNA_ALS2 or LbCpfl +Ribo-crRNA_ALS4).
  • GE components expressed via a binary vector (pZFNnptll) were used as non-GVR controls.
  • DNA level were measured via qPCR using LbCpfl -specific primer at 6 (sample numbers 65, 66, 67, 68, 125, 126, 127, 128, 85, 86, 87, 88, 145, 146, 147, 148) and 12 (sample numbers 69, 70, 71 , 72, 129, 130, 131 , 132, 89, 91 , 92, 149, 150, 151 , 51 , 111 , 112) days post infiltration (dpi). Each bar represents one biological replicate per construct and harvesting time point. Sample 112 (boxed) was used as a reference (set as 1).
  • Figure 12 shows increased DNA levels (from the LbCpfl -encoding gene) in BSCTV-GVRs infiltrated plants compared to the corresponding binary vector controls.
  • Cotyledons of sugar beet seedlings were syringe-infiltrated with GVRs derived from BSCTV (pZFNnptll-BSCTV-Repl), which contain genome editing components (LbCpfl , LbCpfl +tDT-crRNA_ALS2 or LbCpfl +Ribo- crRNA_ALS4).
  • GVRs derived from BSCTV pZFNnptll-BSCTV-Repl
  • GE components expressed via a binary vector were used as non- GVR controls.
  • DNA level were measured via qPCR using LbCpfl -specific primers at 6 (sample numbers 4_9, 4_10, 7_5 - 7_8, 45_5 - 45_8, 1_5 - 1_8, 46_5 - 46_8) and 13 (27_9, 27_10, 7_9,
  • Figure 13 shows indel efficiencies comparing plants infiltrated with the BSCTV_GVRs (A), the BCTIV -GVRs (B) and the respective binary vector controls.
  • A Cotyledons of sugar beet seedlings were syringe-infiltrated with GVRs derived from BSCTV (pZFNnptll-BSCTV-Repl), which contain genome editing components (LbCpfl , LbCpfl +tDT-crRNA_ALS2 or LbCpfl +Ribo- crRNA_ALS4).
  • GE components expressed via a binary vector (pZFNnptll) were used as non- GVR controls.
  • Figure 14 shows sequence sections from GFP (upper panel) and YFP (lower panel), respectively.
  • the nucleotides in boxes are synonymous mutations that are introduced by the donor template into the GFP sequence.
  • the non-synonymous mutations that lead to a conversion of Thr224 into Tyr224 (T224Y) result in conversion of GFP into YFP.
  • Figure 15 shows the repair template (RT) that consists of two homologous arms, which are complementary to the target region in mGFP5-ER.
  • the “core” of the RT includes 23 synonymous mutations, which avoid binding and cutting of the CRISPR complex after RT integration.
  • the “core” includes 3 non-synonymous mutations, which lead to the desired T224Y amino acid exchange.
  • Figure 16 shows the crRNA expression cassette, which is part of the construct pZFNnptll- LbCpf1JDT-crRNA_mGFP#1+2+3_GFRoYFP template (SEQ ID NO: 32).
  • FIG. 18 shows homology-directed repair (HDR) after the induction of CRISPR/Cas-mediated double-stand breaks (DSBs) in GFP transgenic Nicotians benthamiana (16c) using a Beet severe curly top virus replicon (BSCTVRepl) as a delivery vector.
  • HDR homology-directed repair
  • DSBs CRISPR/Cas-mediated double-stand breaks
  • BSCTVRepl Beet severe curly top virus replicon
  • GFP to YFP conversion is shown in B, C and D (marked with arrows) transgenic plants which have been inoculated with pZFN-LbCpf1-tDT-crRNA_mGFP#1+2+3-GFPYFP-RT (B), pZFN-BSCTV-Repl-LbCpf1-tDT-crRNA_mGFP#1+2+3-GFPYFP-RT (C) or pZFN-BSCTVRepl- tDT-crRNA_mGFP#1+2+3-GFPYFP-RT200-Out-lbcpf1 (D), respectively.
  • the present invention is based on the relevant finding that plants known to be recalcitrant to transformation can be conveniently modified via genome editing/engineering (GE) in case new viral replicons are provided, specifically designed and optimized to (i) have a tropism for the relevant plant, (ii) to be safe and efficient in their use and in replication, (iii) have the advantage of being cargos to deliver GE tools, and (iv) have additional advantages in specifically interacting with a plant cell cycle so that desired and site-specific HDR events are strongly favored.
  • GE genome editing/engineering
  • a method for the targeted modification of at least one genomic target sequence in a plant cell preferably from a plant of the family Amaranthacea, preferably a cell of a plant from the genus Beta or Spinacia, more preferably a cell of a plant of the species Beta vulgaris (e.g., sugar beet; SB) or Spinacia oleracea (spinach)
  • the method comprises the following steps: (a) introducing into the cell (i) at least one viral replicon, or a sequence encoding the same, the replicon being preferably constructed based on a Becurtovirus, or a Curtovirus genome, wherein the at least one viral replicon further comprises: (ii) at least one genome editing system comprising at least one nucleic acid guided nuclease, nickase or an inactivated nuclease, or a sequence encoding the same, and optionally, in case a CRISPR system is used, at least
  • a method forthe targeted modification of at least one genomic target sequence in a plant cell preferably from a plant of the family of Solanaceae, preferably a cell of a plant from the genus Nicotiana, more preferably a cell of a plant of the species Nicotiana tabacum or Nicotiana benthamiana
  • the method comprises the following steps: (a) introducing into the cell (i) at least one viral replicon, or a sequence encoding the same, the replicon being preferably constructed based on a Becurtovirus, or a Curtovirus genome, wherein the at least one viral replicon further comprises: (ii) at least one genome editing system comprising at least one nucleic acid guided nuclease, nickase or an inactivated nuclease, or a sequence encoding the same, and optionally, in case a CRISPR system is used, at least one guide molecule, or a sequence
  • the present invention hereby solves the problem to provide methods and systems for increasing the efficiency of targeted modification of a nucleotide sequence of interest in the genome of a plant or a plant cell recalcitrant to modifications with state of the art methods and thus provides efficient and robust methods to regenerate new plants from the transformed plant cells, or plant material.
  • Beta vulgaris plant is not a natural host forthe Mastrevirus BeYDV
  • sugar beet BeYDV-based GVRs do not replicate and cannot interact with host factors as efficient as in model plants, e.g., Nicotiana tabacum or Arabidopsis. Therefore, no suitable sugar beet Geminivirus-based replicons can be obtained starting from Geminiviruses per se having a low tropism or infectious and replicative potential in this commercially relevant plant.
  • Beta vulgaris particularly in sugar beet
  • GVRs derived from Geminiviruses from different viral genera.
  • the present invention thus specifically provides replicons of two viruses for the heterologous expression of genome editing components in Beta vulgaris, namely:
  • BCTIV Beet curly top iran virus (BCTIV, SEQ ID NO: 22), belonging to the genus Becurtovirus;
  • GVRs derived from BSCTV or BCTIV leads to a significantly increased replication intensity in Beta vulgaris leading to high expression levels of the heterologous sequence.
  • This heterologous sequence can for instance encode GE components as well as optionally a repair template and thereby might enhance HDR-mediated genome editing efficiency.
  • GVRs can revert host cells into the S-phase of the cell cycle, which stimulates homology-dependent recombination and thus increases efficiency additionally.
  • Beta vulgaris such as sugar beet, fodder beet, red beet, or chard
  • species like for example Beta vulgaris (such as sugar beet, fodder beet, red beet, or chard) are well known as being very recalcitrant in transgenic modifications.
  • Other dicotyledonous plants in particular plants within the taxonomic family of Amaranthacea.
  • the inventors found that certain of these replicons can have a broad spectrum oftropism for other target plants, e.g., non-crop plants.
  • GVRs Geminivirus replicons
  • sugar beet- infecting viruses is not restricted to sugar beet but can be extended to other non-crop species like tobacco ( Nicotiana benthamiana) belonging to the family Solanaceae).
  • tobacco Nicotiana benthamiana
  • the viral replicons established may additionally be suitable to transform other target plants effectively, particularly those target plants known to be easy or relatively easy to transform or transfect.
  • the target plant to be transformed according to the methods of the present invention is thus selected from a plant belonging to the family Amaranthacea or Solanaceae.
  • the methods disclosed herein are thus genotype-independent based on the preceding efforts to provide new GVRs based on specific (Be-)Curtoviruses.
  • a genomic target sequence of interest according to any of the embodiments described herein may be an endogenous or isolated nucleic acid region of a plant cell or organism including any organellar genomic target sequence.
  • the at least one viral replicon, or a sequence encoding the same may be a replicon being constructed based on a Becurtovirus, or a Curtovirus genome.
  • GVRs Geminivirus-based replicons
  • a viral replicon as used herein thus refers to an expression construct based on a specific naturally occurring virus and comprising a DNA molecule or RNA molecule, or a region of DNA or RNA, that can replicate from a single origin of replication, wherein the viral replicon maintains relevant characteristics of the native virus, in particular regarding the plant tropism and its replication efficiency and further beneficial effects, but is modified to be used as expression construct and does not comprise all virulence factors of the native virus.
  • BCTIV Beet curly top iran virus
  • BSCTV Beet severe curly top virus
  • BCTV Beet curly top virus
  • Curtoviruses are monopartite Geminiviruses that are vectored by leafhoppers, often in a species-specific manner and rather different to Mastreviruses.
  • BCTV encodes usually 7 genes (V1 , V2, V3 on virion-sense strand and Rep, C2, C3 and C4 on the complementary-sense strand) without SIR sequence. Therefore, Becurtovirus and Curtovirus are different in genomic architecture to other Geminivirus replicon systems and thus need certain optimizations to be suitable at all for use as viral replicons, as comparable replicons are not available at date.
  • heterologous expression of nucleic acid material for the purpose of GE is achieved using a specifically designed viral replicon derived from, in particular, a Beta vulgaris infecting Geminivirus (GV), or particularly modified variants of said GV.
  • GV Beta vulgaris infecting Geminivirus
  • modified GV variants have been demonstrated to be suitable for heterologous expression of a nucleotide sequence of interest in planta.
  • the artificially created Geminivirus replicons (GVRs) of the present invention replicate efficiently inside host cells and therefore produce high amounts of replicons, in turn producing high expression of genome editing effector molecules cloned into the respective GVR.
  • this can help to increase the abundance of donor or repair templates available for HDR, thereby considerably enhancing the targeting efficiency and accuracy.
  • GVRs In addition to rolling circle replication, GVRs as studied and constructed herein, replicate inside host cells also via homologous recombination (HR)-dependent replication, which may revert host plant cells to the S-phase.
  • HR homologous recombination
  • the S-phase-like endocycle is favorable for HR if, for example, supplemented with site-specific nucleases and complementary donor template.
  • the viral replicon may be provided as single-component replicon for BCTIV, BSCTV or BCTV.
  • a BCTIV replicon may contain LIR, SIR and Rep, or a BCTV or BSCTV replicon may contain LIR and Rep, but no SIR sequence.
  • the inventors further found that HDR-based GE efficiency can be significantly increased by using the specific (Be-)Curtovirus GVR, as these viral replicons were constructed in a way maintaining their effect of reverting a plant target cell in the S-phase of the cell cycle, which is highly favorable for HDR to occur, and which stimulates HDR- events.
  • a viral replicon according to the present invention may thus be used to revert a host cell of interest into the S-phase and/or to improve HDR-mediated GE efficiency by specifically combining the effects inherent to the new viral replicons of interest as cargo and the GE inserts inserted into these GVR tools.
  • This observation is supported by recent studies done in Arabidopsis thaliana infected with the Geminivirus Euphorbia yellow mosaic virus (EuYMV) (Richter, K. S., et al. (2016). "The recombination mediator RAD51D promotes geminiviral infection.” Virology 493: 113-127.).
  • a viral replicon optimized for plant transformation and based on the relevant findings on new GVRs based on (Be-)curtovirus genomes as disclosed herein wherein the viral replicon may be encoded by a sequence according to SEQ ID NO: 4 or 6, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
  • BCTIV- and BSCTV-GVRs may thus be individually equipped with various GE system components or tools, which are necessary to mediate SDN-1 events and optionally with at least one repair template to mediate SDN-2/3 kind modifications.
  • introduction of at least one viral replicon may be achieved by biological or physical means, including direct application to a cell, transfection, transformation, including transformation by Agrobacterium spp., preferably by Agrobacterium tumefaciens, biolistic bombardment, transfection using chemical agents, including polyethylene glycol transfection, or any combination thereof.
  • the viral replicon may be integrated into a vector suitable for Agrobactehum-me0 ⁇ ate0 plant (cell) transformation such as a binary vector.
  • the viral replicon may be integrated into a vector suitable for transformation via biolistic bombardment; this is not necessarily a binary vector.
  • the at least one viral replicon, or the sequence encoding the same may be constructed based on a Beet curly top iran virus Becurtovirus genome, a Spinach Curly Top Arizona Virus Becurtovirus genome, a Beet severe curly top virus Curtovirus genome, a Beet curly top virus Curtovirus genome, a Beet mild curly top virus Curtovirus genome, or a Spinach severe curly top virus Curtovirus genome, preferably a genome selected from SEQ ID NOs: 22, 23, 27 or 28, or a genome from a homologous virus to the respective sequences belonging to the genus Becurtovirus or Curtovirus, wherein a homologous virus may be understood as a naturally occurring plant infecting virus having a comparable genome architecture as a Becurtovirus or Curtovirus and thus belonging to the same genus of Becurtovirus or Curtovirus, but having certain sequence variations.
  • the at least one viral replicon according to the various aspects and embodiments disclosed herein may be encoded by a sequence according to SEQ ID NO: 4 or 6, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
  • nucleic acid or amino acid sequences Whenever the present disclosure relates to the percentage of the homology or identity of nucleic acid or amino acid sequences to each other over the entire length of the sequences to be compared to each other, wherein these identity or homology values define those as obtained by using the EMBOSS Water Pairwise Sequence Alignments (nucleotide) program (www.ebi.ac.uk/Tools/psa/emboss_water/nucleotide.html) for nucleic acid sequences or the EMBOSS Water Pairwise Sequence Alignments (protein) program (www.ebi.ac.uk/Tools/psa/emboss_water/) for amino acid sequences.
  • any suitable delivery method to introduce the components (i) or (ii) and optionally (iii) according to step (a) of the methods of the present invention into a cell can be applied, depending on the target cell.
  • the term "introducing” as used herein thus implies a functional transport of a biomolecule or genetic construct (DNA, RNA, single- or double-stranded, protein, comprising natural and/or synthetic components, or a mixture thereof) into a cell or into a cellular compartment of interest, e.g.
  • nucleus or an organelle, or into the cytoplasm which allows the transcription and/or translation and/or the catalytic activity and/or binding activity, including the binding of a nucleic acid molecule to another nucleic acid molecule, including DNA or RNA, or the binding of a protein to a target structure within the cell, and/or the catalytic activity of an enzyme such introduced, optionally after transcription and/or translation.
  • a variety of delivery techniques may be suitable according to the methods of the present invention for introducing the components (i) or (ii) and optionally (iii) into a cell, in particular a plant cell, the delivery methods being known to the skilled person, e.g. by choosing direct delivery techniques ranging from polyethylene glycol (PEG) treatment of protoplasts, procedures like electroporation, microinjection, silicon carbide fiber whisker technology, viral vector mediated approaches and particle bombardment.
  • PEG polyethylene glycol
  • a common biological means, and a preferred cargo according to the present invention, is transformation with Agrobacterium spp. which has been used for decades for a variety of different plant materials. Viral vector mediated plant transformation represents a further strategy for introducing genetic material into a cell of interest.
  • the at least one viral replicon comprising the at least one genome editing system may thus be introduced into the cell by transformation or transfection, preferably by Agrobacterium- mediated transformation, biolistic bombardment, micro- or nanoparticle delivery, or by chemical transfection.
  • Specific vectors have been constructed for use according to the present disclosure and optimized for efficiently delivering the viral replicons and the encoded sequences into a cell of interest.
  • At least one viral replicon comprising at least one genome editing system, wherein the replicon is introduced into the cell by Agrobacterium- mediated transformation, wherein the at least one viral replicon is inserted into a binary vector, preferably wherein the binary vector comprising the at least one viral replicon has a sequence of SEQ ID NOs: 5 or 7 to 15, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
  • vectors e.g., artificial vectors suitable as shuttle vectors, so-called because they are able to replicate in multiple hosts, e.g., E. coli and Agrobacterium comprising a transfer DNA (T-DNA) binary system necessary for agrobacterium transformation may be used and are available to the skilled person.
  • T-DNA transfer DNA
  • the methods for the targeted modification of at least one genomic target sequence in a plant cell are used to genome editing (GE) purposes.
  • the GE strategy may be an SDN-1 strategy exclusively introducing GE tools of interest, but no further foreign nucleic acid material.
  • SDN-2/3 strategies additionally introducing at least one repair template ordonortemplate, said terms being used interchangeably herein, may be desired. Both strategies have been shown to be compatible with the specific viral replicons designed.
  • the at least one genome editing system may preferably comprise at least one nucleic acid guided nuclease, nickase or an inactivated nuclease, or a sequence encoding the same, and at least one guide molecule, or a sequence encoding the same.
  • a "(targeted) modification" of at least one genomic target sequence in the context of the present invention as mediated by a GE system of interest refers to any change of a (nucleic acid) sequence that results in at least one difference in the (nucleic acid) sequence distinguishing it from the original sequence.
  • the at least one "(targeted) modification” can be introduced as a consequence of the endogenous repair mechanism (e.g., as a consequence of NHEJ and other known endogenous repair mechanisms) after the at least one GE system introduced in a targeted way was active, or as a direct consequence of the at least one GE system, e.g., a base editor system, or another fusion system, or as a consequence of a repair template (RT) introduced at the site of a single- or double stranded break as introduced by at least one GE system.
  • the genomic target sequence to be modified in a targeted manner can be present in the nuclear, or in the plastid genome, including, but not being limited thereto, the mitochondrial, and the chloroplast genome.
  • genomic target sequence to be modified may be present in a cell as a target nucleic acid sequence present in a cell extra-chromosomally, for example, as autonomously replicating plasmid, or as nucleic acid plasmid transiently introduced into a cell, but not (fully) integrated into the genome of a target cell.
  • a modification can be achieved by insertion or addition of one or more nucleotide(s), or substitution or deletion of one or more nucleotide(s) of the original sequence, e.g., a targeted point mutation as mediated by a singlenucleotide exchange, or any combination of these.
  • a targeted modification can comprise more than one modification mediated simultaneously, for example, more than one point mutation, wherein the individual point mutations can be at least one synonymous mutation (resulting in a difference in the nucleic acid sequence, yet not in an exchange of the encoded amino acid sequence, when the sequence encodes an amino acid sequence) together with at least one non-synonymous mutation.
  • a targeted modification can comprise introducing at least one point mutation (synonymous and/or non-synonymous) together with either one of a deletion and/or an insertion in one and the same experiment, i.e., simultaneously.
  • a targeted modification is introduced using site-specific tools such as at least one nucleic acid guided nuclease, or a variant thereof (including a nickase, or a nuclease-dead variant) which recognizes and/or cuts the target at a specific location. If two or more different guide nucleic acid sequences are used, it is possible to target multiple sites in the genomic target region and introduce “multiple modifications".
  • the “multiple modifications” may also be made, for example, in the same gene, or the “multiple modifications” may be made into regions of the genome not directly connected, e.g., modifications in different chromosomes, and the like.
  • More than one, or “multiple modifications” can be introduced, by way of example, by providing a repair template (RT) harboring all desired modifications in the form of at least one point mutation and, optionally, the capacity to mediate an insertion or deletion in addition to the point mutations.
  • RT repair template
  • more than one guide RNA or crRNA may be used when “multiple modifications" are of interest, or one one guide RNA or crRNA may be used together with a RT carrying more than one modification to be effected.
  • a preferred GE system according to the present disclosure is preferably based on a CRISPR system comprising at least one CRISPR nuclease and at least one guide RNA.
  • a "site-directed nuclease” or “site-specific nuclease” can be a single stranded DNA break (SSB) inducing enzyme or a double stranded DNA break (DSB) inducing enzyme, which preferably recognizes a predetermined site in the genome of targeted cell.
  • SSB single stranded DNA break
  • DSB double stranded DNA break
  • CRISPR nuclease is a specific form of a site-directed nuclease and refers to any nucleic acid guided nuclease, which has been identified in a naturally occurring CRISPR system, which has subsequently been isolated from its natural context, and which preferably has been modified or combined into a recombinant construct of interest to be suitable as tool for targeted genome engineering.
  • Any CRISPR nuclease can be used and optionally reprogrammed or additionally mutated to be suitable for the various embodiments according to the present invention as long as the original wild-type CRISPR nuclease provides for DNA recognition, i.e., binding properties.
  • CRISPR nucleases also comprise mutants or catalytically active fragments or fusions of a naturally occurring CRISPR effector sequences, or the respective sequences encoding the same.
  • a CRISPR nuclease may in particular also refer to a CRISPR nickase or even a nuclease-dead variant of a CRISPR polypeptide having endonucleolytic function in its natural environment.
  • the CRISPR nucleases include CRISPR/Cas systems, including CRISPR/Cas9 systems, CRISPR/Cpfl systems, CRISPR/C2C2 systems, CRISPR/CasX systems, CRISPR/CasY systems, CRISPR/Cmr systems, CRISPR/MAD systems, including, for example, CRISPR/MAD7 systems and CRISPR/MAD2 systems, CRISPR/CasZ systems and/or any combination, variant, or catalytically active fragment thereof.
  • a nuclease may be a DNAse and/or an RNAse, in particular taking into consideration that certain CRISPR effector nucleases have RNA cleavage activity alone, or in addition to the DNA cleavage activity.
  • CRISPR system is thus to be understood as a combination of a CRISPR nuclease or CRISPR effector, or a nickase or a nuclease-dead variant of said nuclease, or a functional active fragment or variant thereof together with the cognate guide RNA (or pegRNA orcrRNA) guiding the relevant CRISPR nuclease.
  • a nucleic acid guided nuclease, or site-specific or site-directed nuclease, of any kind as delivered with a viral replicon according to the present invention thus recognizes a certain protospacer adjacent motif (PAM) at the target site, which is required to be present for the nuclease to cut the target site.
  • PAM protospacer adjacent motif
  • the "PAM specificity" of a nuclease defines, which PAM(s) the nuclease recognizes. For example, certain variations of a PAM or different PAMs may result in cleavage. The different variants or different PAMs may provide a varying degree of nuclease activity at a target site.
  • a nuclease is considered to “recognize” a certain PAM, when the indel percentage at a certain site with the PAM, normalized by transformation efficiency in the system used, is at least 10%, preferably at least 20%, at least 30%, at least 40%, at least 50% or at least 60%.
  • a “higher activity” of a nuclease refers to a higher indel percentage at a certain target site compared to another nuclease.
  • An “indel” refers to an insertion or a deletion of one or more nucleotides at the target site, which is due to site specific nuclease activity at the target site.
  • the at least one nucleic acid guided nuclease, nickase or an inactivated nuclease, or a sequence encoding the same, of the genome editing system may thus be selected from the group consisting of a CRISPR/Cas system, preferably from a CRISPR/Cfp1 system, a CRISPR/MAD7 system, a CRISPR/MAD2 system, a CRISPR/Cas9 system, a CRISPR/CasX system, a CRISPR/CasY system, or a CRISPR/Csm system, or any combination, variant, or catalytically active fragment thereof.
  • a CRISPR/Cas system preferably from a CRISPR/Cfp1 system, a CRISPR/MAD7 system, a CRISPR/MAD2 system, a CRISPR/Cas9 system, a CRISPR/CasX system, a CRISPR/CasY system, or
  • the GE system may be a base editor (BE) system.
  • Base editors including CBEs (base editors mediating C to T conversion) and ABEs (adenine base editors mediating A to G conversion), are powerful tools to introduce direct and programmable mutations without the need for double-stranded cleavage ( Komor ef a/., Nature, 2016, 533(7603), 420-424; Gaudelli et al., Nature, 2017, 551 , 464-471).
  • base editors are composed of at least one DNA targeting module and a catalytic domain that deaminates cytidine or adenine.
  • Base editing does not cut the double-stranded DNA but instead uses the CRISPR targeting machinery to shuttle an additional enzyme to a desired sequence, where it converts a single nucleotide into another.
  • CRISPR targeting machinery to shuttle an additional enzyme to a desired sequence, where it converts a single nucleotide into another.
  • Many genetic traits in plants and certain susceptibility to diseases caused by plant pathogens are caused by a single nucleotide change, so base editing offers a powerful alternative for GE.
  • the method has intrinsic limitations and is said to introduce off-target mutations, which are generally not desired for high precision GE.
  • the GE system may be a Prime Editing system ( Anzalone et al. (2019). Search-and-replace genome editing without double-strand breaks (DSBs) or donor DNA. Nature, 1-1.).
  • Prime Editing (PE) systems steer around the shortcomings of earlier CRISPR based GE techniques by heavily modifying the Cas9 protein and the guide RNA. The altered Cas9 only "nicks" a single strand of the double helix, instead of cutting both.
  • the new guide RNA called a pegRNA (prime editing extended guide RNA), contains an RNA template for a new DNA sequence, to be added to the genome at the target location.
  • the PE system may significantly increase the targeting range of a respective GE system in view of the fact that BEs cannot cover all intended nucleotide transitions/mutations (C®A, C®G, G®C, G®T, A®C, A®T, T®A, and T®G) due to the very nature of the respective systems, and the transitions as supported by BEs may require DSBs in many cell types and organisms.
  • the at least one GE system thus may further comprise at least one PE system and plant host compatible reverse transcriptase, preferably a modified reverse transcriptase, wherein the reverse transcriptase is preferably used in combination with a nucleic acid guided nickase, and/or at least one cytidine or adenine deaminase, preferably wherein the at least one cytidine or adenine deaminase is independently selected from an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase, preferably a rat-derived APOBEC, an activation-induced cytidine deaminase (AID), an ACF1/ASE deaminase, an ADAT family deaminase, an ADAR2 deaminase, or a PmCDAI deaminase, a TadA derived deaminase, and/or
  • APOBEC apolipoprotein B
  • nucleic acid sequence of the GE systems disclosed herein may be “codon optimized” for the codon usage of a plant target cell of interest. This means that the sequence is adapted to the preferred codon usage in the organism that it is to be expressed in, i.e. a “target cell of interest”. If a nucleic acid sequence is expressed in a heterologous system, codon optimization increases the translation efficiency significantly.
  • a GE system in particular a CRISPR system, comprises at least one nucleic acid guided nuclease and at least one "guide molecule” or “guide nucleic acid sequence” (usually called and abbreviated as guide RNA, crRNA, crRNA+tracrRNA, gRNA, sgRNA), which recognizes a target sequence to be cut by the nuclease.
  • the at least one "guide nucleic acid sequence” or “guide molecule” comprises a "scaffold region” and a "target region”.
  • the "scaffold region” is a sequence, to which the nucleic acid guided nuclease binds to form a targetable nuclease complex.
  • the scaffold region may comprise direct repeats, which are recognized and processed by the nucleic acid guided nuclease to provide mature crRNA.
  • a pegRNA may comprise a further region within the guide molecule, the so-called "primer-binding site".
  • the "target region” defines the complementarity to the target site, which is intended to be cleaved.
  • a crRNA as used herein may thus be used interchangeably herein with the term guide RNA in case it unifies the effects of meanwhile well-established CRISPR nuclease guide RNA functionalities.
  • CRISPR nucleases e.g., Cas9
  • Cas9 may be used by providing two individual guide nucleic acid sequences in the form of a tracrRNA and a crRNA, which may be provided separately, or linked via covalent or non-covalent bonds/interactions.
  • the guide RNA may also be a pegRNA of a Prime Editing system as further disclosed above.
  • a “target region/sequence” defines the complementarity to the target site, which is intended to be cleaved
  • a “genomic target region/sequence” is a region in the genome, including the organellar genome, of the target cell, which is to be modified using the GE system with a viral replicon of the present invention.
  • the target region can be an endogenous sequence, e.g. an endogenous target gene, or an isolated nucleic acid region, which is not part of the genome of the target cell but e.g. present on a plasmid or an artificial chromosome.
  • a “repair template” (RT) or “donor template/nucleic acid” represents a single-stranded or double- stranded nucleic acid sequence, which can be provided during any genome editing causing a double-strand or single-strand DNA break to assist the targeted repair of said DNA break by providing a RT as template of known sequence assisting homology-directed repair.
  • the RT may comprise "symmetric or asymmetric homology arms", which provide homology to the sequences flanking the double-strand break introduced by the nuclease and thus promote error-free homology directed repair.
  • the repair template may also comprise at least one chemically modified base or backbone, wherein a "chemically modified base” is present in the repair template, when at least one nucleobase has been modified to carry one or more substituent(s) or label(s) or one or more nucleotide(s) carry a molecule other than a nucleobase instead of a nucleobase.
  • a "chemically modified back bone” is present in the repair template, for example, when the phosphate back bone carries at least one modification such as e.g.
  • a repair template can thus be provided during any genome editing causing a doublestrand and/or single-strand DNA break to assist the targeted repair of said DNA break by providing a RT as template of known sequence assisting homology-directed repair.
  • a repair template can be provided as individual molecule, or a sequence encoding the same, or it may be associated with a guide RNA of a CRISPR nuclease, wherein the association can be of non- covalent nature (complementary base pairing), or the molecules can be physically attached to each other by a covalent bond.
  • the latter embodiment may increase the availability of the at least one repair template of interest at the target site in a genome, where a targeted double- or single- stranded break was introduced which is to be repaired.
  • the at least one repair template may comprise symmetric or asymmetric homology arms and/or the at least one repair template may comprise at least one chemically modified base and/or backbone.
  • the at least one genome editing system may comprise at least one repair template, and wherein the at least one repair template may comprise or encode a double- and/or single-stranded nucleic acid sequence, and/or wherein the at least one repair template may be part of the at least one guide molecule.
  • what is provided may be a plant, plant cell, tissue, organ, or seed obtainable by or obtained by a method according to any of the methods for the targeted modification of at least one genomic target sequence in a plant cell as disclosed herein.
  • the plant material modified in a targeted manner can be used to obtain a whole plant organism, or a part thereof, carrying the desired modification, preferably in an inheritable manner.
  • "Regenerating / regeneration” in this context is to be understood broadly and refers to any kind of in plants, in vitro, or a combined in vivo/ex vivo cultivation of at least one modified plant cell, tissue, organ, or material.
  • Suitable conditions to cultivate plant cells and plant material as modified according to the methods of the present invention in vitro and in vivo are disclosed herein and are known to the skilled person.
  • an inducible promoter system may be used for the expression of the viral replication initiator protein (Rep).
  • inducible systems include but are not limited to inducible promoter systems such as the abscisic acid-inducible promoter (SEQ ID NOs: 1 and 2) or a glucocorticoid receptor-based system inducible by dexamethasone ( Figure 2, SEQ ID NO: 3). Further inducible systems compatible for use in plant cells are known to the skilled person.
  • the methods of the present invention may be performed by transiently introducing at least one viral replicon, or a sequence encoding the same and/or at least one genome editing system comprising at least one nucleic acid guided nuclease, nickase or an inactivated nuclease, or a sequence encoding the same, and at least one guide molecule, or a sequence encoding the same and/or and, optionally at least one repair template, or a sequence encoding the same, whereas in other embodiments, the tools may be introduced in a stable way.
  • a combination of transient and stable transformations is possible in view of the fact that at least two components are inserted into the at least one cell to be modified. "Transient” implies that the tools are only temporarily expressed and afterwards degraded by the cell, whereas “stable” implies that at least one of the tools is integrated into the nuclear and/or organellar genome of the cell to be modified.
  • an expression construct i.e., a nucleic acid cassette comprising several elements fortranscribing and/or translating encoded elements in at least one target cell of interest, which may comprise or encode: (i) at least one viral replicon based on a (Be-)Curtovirus as disclosed according to the various aspects and embodiments herein, and (ii) a vector, preferably a (binary) vector as defined above suitable for Agrobacterium- mediated transformation, and (iii) at least one component of at least one a genome editing system, wherein the genome editing system comprises or encodes: (a) at least one nucleic acid guided or site- specific nuclease, nickase or an inactivated nuclease, or at least one site-directed nuclease, nickase or an inactivated nuclease, and optionally at least one nuclear or organellar localization sequence; and (b) at least one guide molecule for guiding the at least one nucleic
  • the construct comprises or encodes at least one regulatory sequence, wherein the regulatory sequence is selected from the group consisting of a core promoter sequence, a proximal promoter sequence, a cis regulatory sequence, a trans regulatory sequence, a locus control sequence, an insulator sequence, a silencer sequence, an enhancer sequence, a terminator sequence, an intron sequence, and/or any combination thereof.
  • the regulatory sequence is selected from the group consisting of a core promoter sequence, a proximal promoter sequence, a cis regulatory sequence, a trans regulatory sequence, a locus control sequence, an insulator sequence, a silencer sequence, an enhancer sequence, a terminator sequence, an intron sequence, and/or any combination thereof.
  • the regulatory sequence comprises or encodes at least one promoter selected from the group consisting of ZmUbH , BdUbHO, ZmEfl , a double 35S promoter, a rice U6 (OsU6) promoter, a rice actin promoter, a maize U6 promoter, PcUbi4, Nos promoter, AtUbHO, BdEF1 , MeEF1 , HSP70, EsEF1 , MdHMGRI , or a combination thereof.
  • a promoter selected from the group consisting of ZmUbH , BdUbHO, ZmEfl , a double 35S promoter, a rice U6 (OsU6) promoter, a rice actin promoter, a maize U6 promoter, PcUbi4, Nos promoter, AtUbHO, BdEF1 , MeEF1 , HSP70, EsEF1 , MdHMGRI , or a combination thereof.
  • the at least one intron is selected from the group consisting of a ZmUbM intron, an FL intron, a BdUbHO intron, a ZmEfl intron, a AdH1 intron, a BdEF1 intron, a MeEF1 intron, an EsEF1 intron, and a HSP70 intron.
  • the construct comprises or encodes a combination of a ZmUbil promoter and a ZmUbil intron, a ZmUbil promoter and FL intron, a BdUbMO promoter and a BdUbMO intron, a ZmEfl promoter and a ZmEfl intron, a double 35S promoter and a AdH1 intron, or a double 35S promoter and a ZmUbil intron, a BdEF1 promoter and BdEF1 intron, a MeEF1 promoter and a MeEF1 intron, a HSP70 promoter and a HSP70 intron, or of an EsEF1 promoter and an EsEF1 intron.
  • the expression construct may comprise at least one terminator, which mediates transcriptional termination at the end of the expression construct or the components thereof and release of the transcript from the transcriptional complex.
  • the regulatory sequence may comprise or encode at least one terminator selected from the group consisting of nosT, a double 35S terminator, a ZmEfl terminator, an AtSac66 terminator, an octopine synthase (ocs) terminator, or a pAG7 terminator, or a combination thereof.
  • a variety of further suitable promoter and/or terminator sequences for use in expression constructs for different plant cells are well known to the skilled person in the relevant field.
  • NLS/OLS nuclear/organellar localization signal/sequence
  • a target protein usually a GE system effector nuclease, nickase or nuclease-dead variant thereof. Suitable NLS and OLS sequences for different plant cells are described in the literature.
  • the expression construct may additionally comprise or encode at least one self-cleaving ribozyme, preferably at least one hammerhead ribozyme and/or at least one hepatitis-delta virus (HDV) ribozyme and/or at least one artichoke, sunflower or rice HDV-like ribozyme.
  • at least one self-cleaving ribozyme preferably at least one hammerhead ribozyme and/or at least one hepatitis-delta virus (HDV) ribozyme and/or at least one artichoke, sunflower or rice HDV-like ribozyme.
  • HDV hepatitis-delta virus
  • a "self-cleaving ribozyme” is an RNA molecule that is capable to catalyse its own cleavage at a specific site. Upon transcription, self-cleaving ribozymes fold into a specific structure, sometimes requiring the presence of certain metal cations, which induces cleavage of the phosphodiester backbone at a certain position. A number of ribozymes are known, which can be used in a variety of settings.
  • ribozymes may be particularly suitable according to the present disclosure to "activate" encoded GE system components, e.g., various guide RNAs etc., by cleaving and thus activating these crRNAs/pegRNAs/guide RNAs to make them available for interaction with a CRISPR effector protein of interest which is turn targeted to a target site in the plant genome to be modified in a site-directed way.
  • the expression construct may thus also comprise at least one ribozyme, which upon transcription cleaves the (RNA) transcript at one or more predetermined location(s). If placed strategically, the ribozyme(s) release and activate the components of the expression construct from the transcript.
  • the sequence encoding the CRISPR nuclease (or variant thereof) may be flanked by a ribozyme at the 5 ' - and at the 3 ' -end.
  • the expression construct may additionally comprise or encode a sequence for an RNA polymerase II (Pol II) promoter.
  • RNA polymerase II promoters which constitute the majority of the characterized promoters, cannot be directly used for CRISPR guide RNA production in vivo because of: (1) The primary transcripts of RNA polymerase II promoters undergo extensive processing such as 5'-end capping, 3'-end polyadenylation, and splicing out of the introns. Some of the modifications may thus render the designed guide RNA non-functional; and (2) the mature RNA molecules are transported into cytosol, thus they are physically separated from the intended targets that are located in the nucleus (Zhang et al., doi:10.21769/BioProtoc.2148).
  • crRNA excised from a Pol ll-driven mRNA transcript may be used in the certain embodiments in the replicons and/or expression constructs and/or binary vectors disclosed herein to effectively provide said crRNAs as guiding RNAs to the cognate CRISPR effector.
  • a construct or replicon according to the present invention may either comprise at least one self-cleaving ribozyme encoding sequence and at least one RNA pol II promoter, or it may comprise both elements.
  • a DNA-dependent RNA polymerase type II promoter may thus be used, preferably together with at least one sequence encoding at least one self-cleaving ribozyme to further enhance the activation of the GE system components as encoded by a viral replicon and/or an expression construct according to the present invention.
  • Suitable promoters are available in the prior art and can be combined as replicon and/or expression construct element based on the disclosure provided herein.
  • Exemplary viral replicons, expression constructs and vectors combining the above elements in various forms to be suitable for the methods disclosed herein are disclosed in the Examples section and are exemplified in the sequence listing.
  • certain elements of the replicons, expression constructs and/or vectors are readily exchangeable and can (or must) be adapted for use in a plant cell of interest, whereas in particular, the overall structure of the (Be-)Curtovirus replicon is relevant to obtain certain effects as disclosed herein.
  • plant or "plant cell” as used herein refer to a plant organism, a plant organ, differentiated and undifferentiated plant tissues, plant cells, seeds, and derivatives and progeny thereof.
  • Plant cells include without limitation, for example, cells from seeds, from mature and immature embryos, meristematic tissues, seedlings, callus tissues in different differentiation states, leaves, flowers, roots, shoots, male or female gametophytes, sporophytes, pollen, pollen tubes and microspores, protoplasts, macroalgae and microalgae.
  • the different eukaryotic cells for example, animal cells, fungal cells or plant cells, can have any degree of ploidity, i.e. they may either be haploid, diploid, tetraploid, hexaploid or polyploid.
  • the family comprises the genus Beta, comprising Beta vulgaris ssp. vulgaris var. vulgaris (chard), Beta vulgaris ssp. vulgaris var. conditiva (beetroot / red beet), Beta vulgaris ssp. vulgaris var. crassa/alba (fodder beet) and Beta vulgaris subsp. vulgaris var. altissima (sugar beet in a narrower sense), the genus Amaranthus, the genus Chenopodium, including C.
  • the viral replicons identified herein may be optimized for use in a usually recalcitrant plant of the family Amaranthacea, but the replicons may also be suitable for transforming less or not recalcitrant plants as illustrated with the enclosed Examples.
  • a viral replicon as disclosed herein based on a (Be-)Curtovirus or a use of an expression construct as disclosed herein in various aspects and embodiments, for stimulating homology-directed repair by reverting a host plant cell into the S- phase of the cell cycle, and/or for increasing homology-directed repair-based genome editing.
  • This aspect is highly desirable in classical plant breeding as well as plant biotechnology.
  • genetic variation in a specific genomic region, or in a specific nucleotide sequence of interest may be increased, a complete allele or a part like a domain, or an exonic region of a gene of interest may be replaced, or the elimination of undesired genetic components/material closely linked a nucleotide sequence of interest in the genome of a plant (so-called linkage drag), which cannot be easily segregated out by conventional breeding, can be obtained.
  • Example 1 Selection of sugar beet infecting Geminivirus strain for replicon construction
  • Full length clones (FLC) of four SB-infecting Geminivirus strains (BSCTV (Beet severe curly top virus), BCTV (Beet Curly Top Virus), BMCTV (Beet Mild Curly Top Virus), BCTIV (Beet curly top iran virus)) of different genera have been used for Agrobacterium- mediated transformation of sugar beet seedlings to compare viral replication intensity in plants. This initial step was necessary to identify new viruses suitable at all for subsequent viral replicon design in view of the fact that viral replicons for certain dicot target plants like sugar beet and spinach are not readily available and cannot be simply derived from exiting viral replicons having specificity for other target plants.
  • BSCTV Beet severe curly top virus
  • BCTV Beet Curly Top Virus
  • BMCTV Beet Mild Curly Top Virus
  • BCTIV Beet curly top iran virus
  • BSCTV Highest replication intensity was demonstrated for BSCTV, medium replication level was measured for BCTV and BCTIV and relatively low replication level were shown for BMCTV ( Figure 1 A). Based on the experimental results, BSCTV and BCTIV were selected for GVR construction. BSCTV and BCTIV belong to different viral genera than BeYDV.
  • Example 2 Inducible expression of the viral replication initiator protein (Rep) for improved callus development and/or shoot regeneration
  • an inducible promoter system may be used forthe expression of the viral replication initiator protein (Rep).
  • inducible systems include but are not limited to inducible promoter systems such as the abscisic acid-inducible promoter (SEQ ID NOs: 1 and 2) or a glucocorticoid receptor-based system inducible by dexamethasone (Figure 2, SEQ ID NO: 3).
  • the BCTIV full length clone (FLC, SEQ ID NO: 22) was used as starting material.
  • FLC full length clone
  • SEQ ID NO: 22 the BCTIV FLC as a template
  • a PCR-based technique was applied to amplify the viral genome without a 1 ,043 bp fragment which covers the viral ORFs for proteins V1 , V2 and V3.
  • MCS multi(ple) cloning site
  • the final BCTIV replicon thus constructed contains a repeat of long intergenic region (LIR), small intergenic region (SIR) and C1 (Rep) and C2 (TrAP) genes, which play a role in the virus replication and DNA accumulation.
  • the replicon was introduced into the T-DNA of binary vector pZFNnptll via Ncol/Pmel sites (see Figure 3, SEQ ID NO: 5).
  • the BSCTV full length clone (SEQ ID NO: 23) was used as further starting material.
  • a PCR-based technique was applied to amplify the viral genome without a 1 ,002 bp fragment which covers the viral ORFs for proteins R2 and R3 and parts of ORF R1 and R4.
  • MCS multi(ple) cloning site
  • the final BSCTV replicon (SEQ ID NO: 6) contains a repeat of LIR and all complementary sense genes including C1, C2, C3 and C4.
  • the replicon was introduced into the T-DNA of binary vector pZFNnptll via Ncol/Pmel sites (see Figure 4, SEQ ID NO: 7).
  • An expression cassette for GVR-mediated transgene expression (for example 35S::GFP) can be introduced via different cloning sites (for example Spel-sites) present in the MCS of pZFNnptll- BCTIV-Rep/MCS and pZFNnptll-BSCTV-Rep/MCS, respectively (SEQ ID NO: 8 and SEQ ID NO: 9).
  • Genome Editing (GE) components (LbCpfl (SEQ ID NO: 24), crRNA (SEQ ID NOs: 16 to 20), and repair templates (ALS as example, SEQ ID NO: 21)) were introduced into pZFNnptll-BCTIV- Rep/MCS (using SEQ ID NO: 5, producing SEQ ID NOs: 12 and 13) and pZFNnptll-BSCTV- Rep/MCS (using SEQ ID NO: 7, producing SEQ ID NOs: 14 and 15) using the following cloning strategy: first, a pcUbi4::LbCpf1 expression cassette was introduced via XmaUApal sites of the MCS into pZFNnptll-BCTIV-Rep/MCS and via Xma ⁇ /Hind ⁇ into pZFNnptll-BSCTV-Rep/MCS.
  • the d35S::crRNA expression cassette was introduced via Sfi ⁇ sites which are present in the MCS of the viral replicons.
  • the repair template is inserted using a unique Hind ⁇ site.
  • the Hind ⁇ site is located outside of the 3 ' end of the Tpea terminator, which is part of the pcUbi4::LbCpf1 expression cassette. Therefore, the Hind ⁇ site only becomes available after pcUbi4::LbCpf1 expression cassette has been introduced into the viral replicons.
  • Example 5 Binary vector pZFNnptll, GE components (LbCpfl and crRNAs) and Agrobacterium tumefaciens strain
  • the viral replicons are embedded in a binary vector named pZFNnptll.
  • This binary vector includes a nptll resistance cassette for in planta selection.
  • the target gene, the PcUbi4::Cpf1 expression cassette and a crRNA, which targets a target region within the target gene were introduced into the viral replicons.
  • the crRNA is embedded in a Pol ll-driven RNA transcript as described before in Zhong et al., 2017. This transcript includes an mRNA encoding the fluorescent protein tDT ( Figure 5 A).
  • the second crRNA version (as included in SEQ ID NOs: 12 and 14) produces the crRNA from a Pol II promoter using the ribozyme-based strategy (He et al., 2017).
  • the crRNA production unit is composed of three parts: the 5'-end encoding the Hammerhead (HH) ribozyme, the middle part encoding the crRNA, and the 3'-end encoding the Artichoke HDV-like ribozyme ( Figure 5 B).
  • the primary transcripts will undergo self-cleavage catalyzed by the intrinsic nuclease activities of the ribozymes to release the mature and desired crRNA.
  • the binary vectors were transformed into agrobacterium strain AGL1-1. Notably, other agrobacterium strains available may be used as well.
  • GFP green fluorescent protein
  • transcription levels were compared between a BCTIV-based replicon (SEQ ID NO: 8), a BSCTV-based replicon (SEQ ID NO: 9), a BeYDV-based replicon (SEQ ID NO: 10) and a standard T-DNA vector without viral replicon elements (SEQ ID NO: 11). Therefore, an expression cassette of 1 ,843 bp in size, which expresses the GFP under the 35S promoter, was cloned into GVRs derived from BSCTV, BCTIV and BeYDV.
  • the binary vector pZFNnptll which includes the 35S::GFP expression cassette, was included as a non-GVR control.
  • the constructs were infiltrated into cotyledons of sugar beet seedlings or tobacco leaves and GFP transcript levels were measured using qRT-PCR (reference gene for sugar beet: GAPDH) after four and six days of infiltration in sugar beet ( Figure 6) and after six days of infiltration in tobacco ( Figure 7).
  • the viral replicons derived from BSCTV and BCTIV clearly outperform BeYDV-GVR and the binary vector control in term of heterologous gene expression.
  • GFP transcript level are strongly increased when expressed by BSCTV or BCTIV-based GVRs compared to the binary vector control and BeYDV-GVR ( Figure 6).
  • BCTIV-GVR and BSCTV-GVR express the GFP gene at a significantly higher levels compared to the binary vector control and BeYDV-GVR at 6 dpi ( Figure 7).
  • GVRs derived from sugar beet-infecting viruses is not restricted to sugar beet but can be extended to other non-crop species like tobacco.
  • SSN nucleic acid guided site-specific nuclease
  • SDN-1 cognate crRNA
  • a PcUbi4::Cpf1 expression cassette of ⁇ 5.2 kb in size and/or a d35S::crRNA expression cassette of 1 .1-1 .7 kb in size which targets one of five different target regions within the target gene acetolactate synthase (BvALS, SEQ ID NOs: 16 to 20) were introduced into the BCTIV-replicon and the BSCTV-replicon ( Figure 5) to mediate genome editing in a sugar beet target gene.
  • BvALS acetolactate synthase
  • Agrobacterium- mediated infiltration of sugar beet (SB) seedlings with BCTIV-derived GVRs revealed that in comparison to the binary vector controls, LbCpfl transcription (Figure 8 shows RT-PCR results) and DNA levels ( Figure 9 shows qPCR results) are strongly increased when expression is mediated by BCTIV-GVR.
  • LbCpfl transcription Figure 8 shows RT-PCR results
  • DNA levels Figure 9 shows qPCR results
  • BCTIV-GVR including only the pcUbi4:: LbCpfl expression cassette of ⁇ 5.2 kb in size shows a higher transcript level than the BCTIV-GVRs including both the LbCpfl and the crRNA expression cassettes.
  • Example 8 Expression of GE components (SDN-2/3)
  • SSN + crRNA + repair template (SDN-2/3) approaches can be tested with the above viral replicons and binary vector systems established.
  • HDR-based genome editing efficiencies in sugar beet are increased by GVR-mediated delivery of (i) site-specific nucleases (SSNs) from the CRISPR/Cas type, (ii) sgRNA and (iii) a repair template (e. g. SEQ ID NO: 21 for BvALS,
  • the GVR is equipped with a repair template, which will be integrated into the genomic target region during homologous recombination. Since GVRs replicate efficiently inside host cells they produce high amounts of replicons, in turn producing high expression of SSNs and increasing the abundance of donor or repair templates available for HDR, thereby considerably enhancing the targeting efficiency. Therefore, BSCTV- and BCTIV-GVR constructs are equipped with the HDR-based GE components LbCpfl , the crRNA and the repair template. The constructs are used for agrobacterium-mediated infiltration of sugar beet leaf explants.
  • Leaf samples are taken 10 days post infiltration (dpi) and they are used for DNA extraction and subsequent NGS analysis to quantify efficiencies of HDR events.
  • Example 9 GVR-based vector system for HDR-based GE in tobacco
  • GVR-based GE Efficiency of GVR-based GE was tested in the transgenic tobacco line Nicotians benthamiana 16c (Philips et al., 2017), which expresses an optimized Aequorea victoria green fluorescent protein (mGFP5-ER, codon optimized and equipped with additional regulatory sequences for the expression in plants (Philips et al., 2017)) constitutively.
  • a single amino acid exchange (T224Y) in GFP causes an altered light emission spectrum that turns GFP to YFP (yellow fluorescent protein (Wachter et al. 1998; Figure 14, SEQ ID NO: 29 and SEQ ID NO: 30)).
  • the repair template that enables the GFP to YFP mutation consists of two homologous arms, which are complementary to the target region in mGFP5-ER. Two nucleotides within the homologous arms were adjusted, to remove Bsal and Bbs ⁇ restriction sites (Figure 15).
  • the “core” of the RT ( Figure 15, lower panel) includes 23 synonymous mutations, which avoid binding and cutting of the CRISPR complex after RT integration.
  • the “core” includes 3 non-synonymous mutations (dark grey), which lead to the desired T224Y amino acid exchange ( Figure 15, SEQ ID NO: 31).
  • a binary vector, which does not include viral replicon sequences was used as non GVR control.
  • the binary vector includes expression cassettes for nptll, LbCpfl and tDT-crRNA_mGFP#1 +2+3, which are directed against three target regions in mGFP5 ( Figure 15, SEQ ID NO: 32).
  • the crRNA is embedded in a Pol ll-driven RNA transcript as described by Zhong et al., 2017.
  • the binary vector includes the repair template described in Figure 15.
  • a binary vector which includes a nptll expression cassette and a viral replicon sequence derived from Beet severe curly top virus (BSCTV, SEQ ID NO: 33).
  • LbCpfl and tDT- crRNA_mGFP#1+2+3 are located within the viral replicon sequence expression cassettes.
  • the crRNA tDT-crRNA_mGFP#1 +2+3 are directed against three target regions in mGFP5-ER ( Figure 16) and embedded in a Pol ll-driven RNA transcript as described by Zhong et al., 2017.
  • the viral replicon includes the repair template described in Figure 15.
  • a binary vector which includes a nptll expression cassette and a viral replicon sequence derived from Beet severe curly top virus (BSCTV, SEQ ID NO: 34).
  • BSCTV Beet severe curly top virus
  • the expression cassette for a tDT- crRNA_mGFP#1 +2+3 ( Figure 16) and the repair template ( Figure 15) are located within the viral replicon.
  • the GVR construct is characterized by an LbCpfl expression cassette that is located outside the viral replicon, but still within the T-DNA.
  • each crRNA was cloned separately in the binary vector pZFNnptll-LbCpfl . Leaves of Nicotiana benthamiana 16c were syringe-infiltrated with these constructs and leaf samples were harvested 3 dpi and 7 dpi for DNA extraction. SDN-1 efficiencies were quantified by ddPCR. Functionality of all three crRNAs was confirmed ( Figure 17). At 7 dpi all selected crRNAs showed considerable SDN-1 efficiency (4-14%).
  • Leaves of Nicotiana benthamiana 16c transgenic line have been syringe infiltrated with either pZFNnptll/LbCpf1_tDT-crRNA_mGFP#1 +2+3_GFPtoYFP template (SEQ ID NO: 32), pZFNnptll- BSCTV-Repl/LbCpf1_tDT-crRNA_mGFP#1+2+3_GFPtoYFP_template (SEQ ID NO: 33) or pZFNnptll-BSCTV-Repl/tDT-crRNA-mGFPm+2+Z-RTGFPtoYFP200-Out LbCpfl (SEQ ID NO: 34).

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Abstract

The present invention relates to the technical field of targeted modification of a nucleotide sequence of interest in the genome of a plant. More particularly, the invention relates to the provision of new viral replicons for delivering genome editing tools to plant cells recalcitrant for transformation for, in particular, achieving homology-directed repair (HDR)-mediated genome editing suitable for, inter alia, plant cells or plants of the species of the family Amaranthacea, preferably a cell of a plant from the genus Beta or Spinacia, and/or species of the family Solanacea, particularly from the genus Nicotiana. The invention thus describes a method for increased efficiency of genome editing, in particular HDR-mediated genome editing, in a plant or a plant cell by providing specific viral replicons facilitating the modification of transformation recalcitrant plants and plant cells.

Description

(Be-)Curtovirus replicon-mediated genome editing in plants
Technical Field
The present invention relates to the technical field of targeted modification of a nucleotide sequence of interest in the genome of a plant. More particularly, the invention relates to the provision of new viral replicons for delivering genome editing tools to plant cells recalcitrant for transformation for, in particular, achieving homology-directed repair (HDR)-mediated genome editing suitable for, inter alia, plant cells or plants of the species of the family Amaranthacea, preferably a cell of a plant from the genus Beta or Spinacia, and/or species of the family Solanacea, particularly from the genus Nicotiana. The invention thus describes a method for increased efficiency of genome editing, in particular HDR-mediated genome editing, in a plant or a plant cell by providing specific viral replicons facilitating the modification of transformation recalcitrant plants and plant cells.
Background of the Invention
While for many crops the use of CRISPR-based genome editing (GE) is described already, there are several crop plants for which only little is known about the successful and efficient methods for such genome editing, in particular complex modifications like HDR-mediated genome editing. For instances, a plant of the species Beta vulgaris (such as, for example, sugar beet, fodder beet, red beet, or chard) is well known as being very recalcitrant in transgenic modifications. There are only very few established methods for transformation, mainly based on Agrobacterium- mediated transformation, which were established for recalcitrant plants, however, with a high level of genotype dependency.
The common way to modify a nucleotide sequence of interest in the genome of e.g. sugar beet is to stably transform plants via Agrobacterium- mediated delivery of a T-DNA vector expressing a nucleotide sequence of interest, which may be a gene conferring an important trait or a gene silencing cassette. Particularly, in sugar beet stable transformation and subsequent regeneration of transgenic plants is amongst the aforementioned technical hurdles extremely labor-intensive, time-consuming and cost-intensive. Moreover, the Agrobacterium- mediated construct insertion into the plant genome does not occur in a targeted way, but randomly which is not desired for obtaining a predictable GE outcome. Therefore, positional effects appear frequently. In contrast to that, efficient GE mediated by site-specific nucleases might allow targeted modification of a nucleotide sequence in the genome of sugar beet and it simultaneously may expand the potential applications, but this requires the provision of new tools for providing the relevant GE tools to a recalcitrant, i.e. , difficult to transform or transfect, target plant.
Technically the most difficult and challenging way of targeted genome editing in any crop is the HDR-mediated approach. This approach allows targeted insertion of larger nucleotide molecules, e.g. entire genes, regulatory elements or the replacement of one allele for another which for example carries one or more individual amino acid substitution. Previous studies in other crops revealed that HDR-mediated genome editing in plants encounters generally two main barriers: 1) efficient HDR-mediated genome modification requires a large amount of exogenous repair template to outcompete the sister chromatid, which is usually used as repair template under natural conditions; 2) when transforming terminally differentiated/non-dividing cells, the pathway for HDR may not be available.
Both requirements for efficient HDR-mediated genome modification cannot be reached by conventional DNA delivery methods like (binary) vectors or other physical delivery. Zaidi & Mansoor (2017) ( Viral vectors for plant genome engineering. Frontiers in plant science, 8, 539) reviewed the potential use of Geminivirus replicons (GVRs) as suitable delivery tools for GE components to induce HDR. Until today, however, GVRs have been used only in plants which are known to be easy transformable and regenerable like Arabidopsis thaliana, Solanum lycopersicum, Nicotiana tabacum, Solanum tuberosum, Nicotiana benthamiana, Triticum aestivum and Oryza sativa. Additionally, a further limitation is the fact that viruses have a very specific plant tropism and the number of Geminiviruses tested positively for transformation purposes yet are very limited.
Zaidi & Mansoor report on three different virus strains, namely BeYDV, CaLCuV and WDV. All these viruses, as naturally occurring, have a specific genome architecture and are not specific in tropism for recalcitrant plants like Beta vulgaris, or other plants in the family Amaranthaceae. The authors highlighted particularly that regenerating plants transformed with Geminivirus vectors has generally proven extremely difficult, mainly because Geminivirus proteins interact with several cellular proteins to facilitate viral replication that in turn compromises the integrity of host cell. Therefore, establishing new viral replicons based on DNA viruses is usually associated with several hurdles to be overcome and a suitable replicon based on a new virus has to be constructed case-by-case taking into consideration the genome structure and pathogenicity mechanism of the virus and the target plant(s) of interest to be transformed.
Lozano-Duran (2016) ( Geminiviruses for biotechnology: the art of parasite taming. New Phytologist, 210(1), 58-64.) indicated that another pleiotropic effect of constitutive replicon expression might be an increase in off-target modifications, generally not desired for precise GE in plants.
EP 2 861 737 B1 disclosed a method to increase the efficiency of GE events in plants, which are based on homology-directed repair. However, the only virus used is the Mastrevirus bean yellow dwarf virus (BeYDV) and, again, only plant species which are known to be susceptible for genetic modification and simple to recover, namely Arabidopsis thaliana and Nicotiana tabacum, are used. Therefore, the approach disclosed is not suitable for efficiently transforming and modifying recalcitrant dicotyledonous plants, as already transformation with the disclosed viruses may be associated with problems due to incompatibility of the relevant host to be transformed and later the host genome to be modified. Since a Beta vulgaris plant is not a natural host for the Mastrevirus BeYDV, in sugar beet BeYDV- based GVRs cannot replicate and interact with host factors as efficient as in the model plants Nicotiana tabacum or Arabidopsis. Moreover, the family of Geminiviridae is highly diverse in structure and function which otherwise necessarily entails that findings for one specific virus from a genus or species attributed to the Geminiviridae family cannot easily be applied to another genus due to large differences in genome organization and pathology.
For example, Bean Yellow Dwarf Virus (BeYDV) which has been used for genome editing ( Baltes et al. 2014. DNA Replicons for Plant Genome Engineering. The Plant Cell 26 (1):151-163. doi:10.1105Apc.113.119792.) is a member of Mastreviruses. The genome of this Geminivirus encodes four genes (Rep, RepA, V1 and V2) and contains a long internal region (LIR) and a short internal region (SIR). Baltes et al. kept the LIR, SIR and Rep and RepA sequences in the BeYDV replicon.
In contrast, Beet curly top iran virus (BCTIV) is a new and not yet detailed studied member of Becurtoviruses with a unique nanonucleotide in the LIR and a rather different member of the family Geminiviridae. This virus encodes five genes (V1 , V2 and V3 on the virion-sense strand and Rep and C2 genes on the complementary-sense strand). The genome sequence and structure of the virion-sense stand of Becurtoviruses is similar to Curtoviruses while the complementary-sense strand has similarity to the Mastreviruses.
Beet severe curly top virus (BSCTV) and Beet curly top virus (BCTV) are members of Curtoviruses. Curtoviruses are monopartite Geminiviruses that are vectored by leafhoppers, often in a species-specific manner and rather different to Mastreviruses. BCTV encodes usually 7 genes (V1 , V2, V3 on the virion-sense strand and Rep, C2, C3 and C4 on the complementary- sense strand) without SIR sequence.
Still, at date little is known about (Be-)Curtovirus replicon architecture allowing efficient strategies for replication and expression of GE tools and having a tropism for recalcitrant plants, e.g. in the family Amaranthaceae.
Thus, there is a great need of providing new viral replicons for safely and efficiently transforming recalcitrant plants and new methods for HDR-mediated modification of nucleic acids of interest in particular for such plant species, which are recalcitrant for genetic modifications and, wherein the methods should have a broad genotype applicability as well as applicability for those genotypes, for which presently few or no commercially interesting transformation techniques are available.
Summary of the Invention
To address the above objectives, the present invention thus provides, in a first aspect, a method for the targeted modification of at least one genomic target sequence in a plant cell preferably from a plant of the family Amaranthaceae or Solanaceae, preferably a cell of a plant from the genus Beta or Spinacia or Nicotiana, more preferably a cell of a plant of the species Beta vulgaris, Spinacia oleracea, Nicotiana benthamiana or Nicotiana tabacum, wherein the method comprises the following steps: (a) introducing into the cell (i) at least one viral replicon, or a sequence encoding the same, the replicon being preferably constructed based on a Becurtovirus, or a Curtovirus genome, wherein the at least one viral replicon further comprises: (ii) at least one genome editing system comprising at least one nucleic acid guided or site-specific nuclease, nickase or an inactivated nuclease, or a sequence encoding the same, and optionally, in the case a CRISPR system is used, at least one guide molecule, or a sequence encoding the same; (iii) and, optionally at least one repair template, or a sequence encoding the same; (b) cultivating the cell under conditions allowing the expression and/or assembly of the at least one genome editing system and optionally the at least one guide molecule and/or optionally the at least one repair template; and (c) obtaining at least one modified cell; and (d) optionally obtaining at least one plant, plant tissue, organ, or seed regenerated from the at least one modified cell.
In a further aspect, a plant, plant cell, tissue, organ, or seed obtainable by or obtained by a method according to the first aspect is provided.
In yet another aspect, a viral replicon for plant transformation is provided, wherein the viral replicon is encoded by a sequence according to SEQ ID NO: 4 or 6, or a sequence having at least sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
In yet a further aspect, an expression construct is provided comprising or encoding: (i) at least one viral replicon as defined in the above aspect and (ii) a vector, preferably a binary vector, more preferably a binary vector as defined in the above aspect, and (iii) at least one component of at least one genome editing system, wherein the genome editing system comprises or encodes: (a) at least one nucleic acid guided or site-specific nuclease, nickase or an inactivated nuclease, or at least one site-directed nuclease, nickase or an inactivated nuclease, and optionally at least one nuclear or organellar localization sequence; and optionally (b) at least one guide molecule for guiding the at least one nucleic acid guided nuclease, nickase or an inactivated nuclease and optionally for activating at least one reverse transcriptase; and (c) optionally at least one repair template; and (d) optionally at least one reverse transcriptase; and (iv) wherein the expression construct comprises or encodes at least one regulatory sequence, wherein the regulatory sequence is selected from the group consisting of a core promoter sequence, a proximal promoter sequence, a cis regulatory sequence, a trans regulatory sequence, a locus control sequence, an insulator sequence, a silencer sequence, an enhancer sequence, a terminator sequence, an intron sequence, and/or any combination thereof.
In a further aspect, a use of a viral replicon optimized in tropism for a plant or plant cell of interest, or a use of an expression construct according to the above aspect is provided, for stimulating homology-directed repair by reverting a host plant cell into the S-phase of the cell cycle, and/or for increasing homology-directed repair-based genome editing.
Brief description of Drawings
Figure 1 shows viral DNA levels of full-length clones (A) and replicons (B). Figure 1 A shows virus accumulation in sugar beet (SB) cotyledons six days post inoculation. Cotyledons of sugar beet seedlings were syringe-infiltrated with full-length clones of different SB- infecting Geminiviruses (BSCTV, BCTV, BMCTV, BCTIV). DNA levels were determined via qPCR six days post infiltration (dpi). Four biological replicates were tested per construct. Error bars represent standard deviations. Figure 1 B shows replication intensities of GVRs in sugar beet. Cotyledons of sugar beet seedlings were syringe-infiltrated with GVRs derived from BSCTV (BSCTVReplM), BCTIV (BCTIVReplM) or BeYDV (LSL). DNA levels were determined via qPCR ten days post infiltration using virus specific primers. A reference gene from Beta vulgaris was used for normalization. Three biological replicates were tested per replicon. Error bars represent standard deviations.
Figure 2 shows a schematic view of the dexamethasone (DEX)-inducible promoter system.
Figure 3 shows a schematic view of the binary vector pZFNnptll containing the BCTIV-based replicon (BCTIV-Rep/MCS in pZFNnptll).
Figure 4 shows a schematic view of the binary vector pZFNnptll containing the BSCTV-based replicon (BSCTV-Rep/MCS in pZFNnptll).
Figure 5 shows the design of the binary vectors used for in planta proof of LbCpfl mediated genome editing. (A) The LbCpfl _tDTcrRNA_ALS#1 (SEQ ID NO: 25) construct harbors expression cassettes for nptll, LbCpfl and crRNA_ALS#1 , which is directed against the target region BvALS#1 . The crRNA is embedded in a Pol ll-driven RNA transcript as described by Zhong et al„ 2017 (doi: 10.1038/nchembio.2410). (B) The LbCpfl _RibocrRNA_ALS#1 (SEQ ID NO: 26) construct harbors expression cassettes for nptll, LbCpfl and a crRNA_ALS#1 , which is directed against the target region BvALS#1. The crRNA is produced from a Pol II promoter using the ribozyme-based strategy.
Figure 6 shows GFP transcript levels in sugar beet. In sugar beet BCTIV-GVR and BSCTV-GVRs outperform BeYDV-GVR in terms of heterologous gene expression. Cotyledons of sugar beet seedlings were syringe-infiltrated with GVRs derived from BeYDV (LSLR), BCTIV (BCTIVReplM) or BSCTV (BSCTVReplM), which contain a 35S::GFP expression cassette. 35S::GFP expressed via a binary vector (pZFN-GFP) was used as non-GVR control. Transcript levels of GFP were measured via qRT-PCR four days (A) or six days (B) post infiltration. GFP expression was normalized against a reference gene from sugar beet. Three biological repeats were tested. Error bars represent standard deviations.
Figure 7 shows GFP transcript levels in Nicotiana benthamiana. Nicotiana benthamiana leaves were syringe-infiltrated with GVRs derived from BeYDV (LSLR), BCTIV (BCTIVReplM) or BSCTV (BSCTVReplM), which contain a 35S::GFP expression cassette. 35S::GFP expressed via a binary vector (pZFN-GFP) was used as non-GVR control. Transcript level of GFP were measured via qRT-PCR six days post infiltration. GFP expression was normalized against a reference gene from N. benthamiana. Three biological replicates were tested. Error bar represent standard deviations. Figure 8 shows that transcription of LbCpfl is increased in BCTIV-GVRs compared to the corresponding binary vector controls. Figure 8/1 shows relative transcription levels for the LbCpf1_tDTcrRNA construct. The samples with the numbers 21 , 22, 23, 24, 41 , 42, 43, 44, 101 , 102, 103, 104, 81 , 82, 83, 84, 141 , 142, 143 and 144 represent relative transcription levels zero days post infiltration (dpi). The samples with the numbers 25, 26, 27, 28, 45, 46, 47, 48, 105, 106, 107, 108, 85, 86, 87, 88, 145, 146, 147, 148 represent relative transcription levels 6 dpi. The samples 29, 30, 31 , 32, 49, 50, 51 , 52, 109, 110, 111 , 112, 89, 90, 91 , 92, 149, 150, 151 , and 152 represent relative transcription levels 12 dpi. Sample 108 (boxed) was used as a reference (set as 1). Figure 8/2 shows relative transcription levels for the LbCpfl _Ribo_crRNA construct. The samples with the numbers 21 , 22, 23, 24, 41 , 42, 43, 44, 101 , 102, 103, 104, 61 , 62, 63, 64, 121 , 122, 123 and 124 represent relative transcription levels 0 dpi. The samples with the numbers 25, 26, 27, 28, 45, 46, 47, 48, 105, 106, 107, 108, 65, 66, 67, 68, 125, 126, 127, 128 represent relative transcription levels 6 dpi. The samples 29, 30, 31 , 32, 49, 50, 51 , 52, 109, 110, 111 , 112, 69, 70, 71 , 72, 129, 130, 131 , and 132 represent relative transcription levels 12 dpi. Sample 108 (boxed) was used as a reference (set as 1).
Figure 9 shows transcription of LbCpfl is increased in plant tissue infiltrated with BSCTV-GVRs compared to the corresponding binary vector controls. Cotyledons of sugar beet seedlings were syringe-infiltrated with GVRs derived from BSCTV (pZFNnptll-BSCTV-Repl), which contain genome editing components (LbCpfl or LbCpfl +crRNA). GE components expressed via a binary vector (pZFNnptll) were used as non-GVR controls. Transcript levels of LbCpfl were measured via qRT-PCR zero (sample numbers: 1 , 2, 3), six (sample numbers: 5, 6, 7) and 13 (sample numbers: 9, 10, 11) days post infiltration (dpi). Three biological replicates per construct and harvesting time point were tested. Figure 9 A and 9 B show the same data, but the x-axes differ to show in B that LbCpfl transcript level mediated by the binary vector controls are expressed at a very low level compared to the BSCTV-GVR constructs.
Figure 10 shows the repair template for BvALS. Schematic view of the double-stranded repair template for introducing an amino acid exchange (Trp-Leu) in BvALS, which confers herbicide resistance towards sulfuronylurea. Templates: Double stranded DNA, Core template = 144 bp, Whole core template mutated with 44 SNPs creating silent mutations + 1 sense mutation conferring herbicide resistance against sulfonylurea, Homologous arms: 2x100 bp or 2x500 bp (or 2x1000 bp).
Figure 11 shows increased DNA levels (from the LbCpfl -encoding gene) in BCTIV-GVRs infiltrated plants compared to the corresponding binary vector controls at 6 dpi and 12 dpi, respectively. Cotyledons of sugar beet seedlings were syringe-infiltrated with GVRs derived from BCTIV (pZFNnptll-BCTIV-Repl), which contain genome editing components (LbCpfl , LbCpfl +tDT-crRNA_ALS2 or LbCpfl +Ribo-crRNA_ALS4). GE components expressed via a binary vector (pZFNnptll) were used as non-GVR controls. DNA level were measured via qPCR using LbCpfl -specific primer at 6 (sample numbers 65, 66, 67, 68, 125, 126, 127, 128, 85, 86, 87, 88, 145, 146, 147, 148) and 12 (sample numbers 69, 70, 71 , 72, 129, 130, 131 , 132, 89, 91 , 92, 149, 150, 151 , 51 , 111 , 112) days post infiltration (dpi). Each bar represents one biological replicate per construct and harvesting time point. Sample 112 (boxed) was used as a reference (set as 1).
Figure 12 shows increased DNA levels (from the LbCpfl -encoding gene) in BSCTV-GVRs infiltrated plants compared to the corresponding binary vector controls. Cotyledons of sugar beet seedlings were syringe-infiltrated with GVRs derived from BSCTV (pZFNnptll-BSCTV-Repl), which contain genome editing components (LbCpfl , LbCpfl +tDT-crRNA_ALS2 or LbCpfl +Ribo- crRNA_ALS4). GE components expressed via a binary vector (pZFNnptll) were used as non- GVR controls. DNA level were measured via qPCR using LbCpfl -specific primers at 6 (sample numbers 4_9, 4_10, 7_5 - 7_8, 45_5 - 45_8, 1_5 - 1_8, 46_5 - 46_8) and 13 (27_9, 27_10, 7_9,
7 _ 10 _ 7 _ 12, 45_9 - 45_12, 1_9 - 1_12, 46_9_46_12) days post infiltration (dpi). Each bar represents one biological replicate per construct and harvesting time point. Sample 1_9 (boxed) was used as a reference (set as 1).
Figure 13 shows indel efficiencies comparing plants infiltrated with the BSCTV_GVRs (A), the BCTIV -GVRs (B) and the respective binary vector controls. (A) Cotyledons of sugar beet seedlings were syringe-infiltrated with GVRs derived from BSCTV (pZFNnptll-BSCTV-Repl), which contain genome editing components (LbCpfl , LbCpfl +tDT-crRNA_ALS2 or LbCpfl +Ribo- crRNA_ALS4). GE components expressed via a binary vector (pZFNnptll) were used as non- GVR controls. Indel efficiencies were determined via ddPCR from leaf samples harvested 13 days post infiltration (dpi). Error bars represent standard deviation. n=2 for constructs lacking a crRNA (non-edited controls); n=3-4 for constructs harboring a crRNA. (B) Cotyledons of sugar beet seedlings were syringe-infiltrated with GVRs derived from BCTIV (pZFNnptll-BCTIV-Repl), which contain genome editing components (LbCpfl , LbCpfl +tDT-crRNA_ALS2 or LbCpfl +Ribo- crRNA_ALS4). GE components expressed via a binary vector (pZFNnptll) were used as non- GVR controls. Indel efficiencies were determined via NGS analysis from leaf samples harvested 12 days post infiltration (dpi). Error bars represent standard deviation. n=2 for constructs lacking the crRNA (non-edited controls); n=3-4 for constructs harboring a crRNA.
Figure 14 shows sequence sections from GFP (upper panel) and YFP (lower panel), respectively. The nucleotides in boxes are synonymous mutations that are introduced by the donor template into the GFP sequence. The non-synonymous mutations that lead to a conversion of Thr224 into Tyr224 (T224Y) result in conversion of GFP into YFP.
Figure 15 shows the repair template (RT) that consists of two homologous arms, which are complementary to the target region in mGFP5-ER. The “core” of the RT includes 23 synonymous mutations, which avoid binding and cutting of the CRISPR complex after RT integration. In addition, the “core” includes 3 non-synonymous mutations, which lead to the desired T224Y amino acid exchange.
Figure 16 shows the crRNA expression cassette, which is part of the construct pZFNnptll- LbCpf1JDT-crRNA_mGFP#1+2+3_GFRoYFP template (SEQ ID NO: 32).
Figure 17 shows SDN-1 editing efficiency in infiltrated tobacco leaves. Nicotiana benthamiana leaves were syringe-infiltrated with pZFNnptll/LbCpf1_tDT-crRNA_mGFP#1 , #2 or #3. pZFNnptll/LbCpfl without any crRNAs was used as a negative control. Indel efficiency was determined via ddPCR from leaf samples harvested 3 and 7 days post infiltration (dpi). Error bars represent standard deviation (n=3).
Figure 18 shows homology-directed repair (HDR) after the induction of CRISPR/Cas-mediated double-stand breaks (DSBs) in GFP transgenic Nicotians benthamiana (16c) using a Beet severe curly top virus replicon (BSCTVRepl) as a delivery vector. Epifluorescence of leaf tissues are shown after excitation with 488-nm laser light for green fluorescent protein (GFP) in the top right panel and 514-nm laser light (top left panel) in the spectral un-mixing for the yellow fluorescent protein (YFP) using confocal laser microscopy. For each treatment a bright field of a leaf (bottom left) and a merge merged image (bottom right) is shown. Images were taken at 7 days after agroinoculation. pZFN-BSCTVRepl-LbCpfl (A) which contains only the nuclease but not the crRNA and donor repair template was used as a control. Conversion of transgene GFP to YFP by homologous recombination occurs after changing a single amino acid mutation (Thr224Tyr) in GFP. GFP to YFP conversion is shown in B, C and D (marked with arrows) transgenic plants which have been inoculated with pZFN-LbCpf1-tDT-crRNA_mGFP#1+2+3-GFPYFP-RT (B), pZFN-BSCTV-Repl-LbCpf1-tDT-crRNA_mGFP#1+2+3-GFPYFP-RT (C) or pZFN-BSCTVRepl- tDT-crRNA_mGFP#1+2+3-GFPYFP-RT200-Out-lbcpf1 (D), respectively.
Brief description of Sequences
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Detailed Description
The present invention is based on the relevant finding that plants known to be recalcitrant to transformation can be conveniently modified via genome editing/engineering (GE) in case new viral replicons are provided, specifically designed and optimized to (i) have a tropism for the relevant plant, (ii) to be safe and efficient in their use and in replication, (iii) have the advantage of being cargos to deliver GE tools, and (iv) have additional advantages in specifically interacting with a plant cell cycle so that desired and site-specific HDR events are strongly favored.
In a first aspect, a method for the targeted modification of at least one genomic target sequence in a plant cell preferably from a plant of the family Amaranthacea, preferably a cell of a plant from the genus Beta or Spinacia, more preferably a cell of a plant of the species Beta vulgaris (e.g., sugar beet; SB) or Spinacia oleracea (spinach) may be provided, wherein the method comprises the following steps: (a) introducing into the cell (i) at least one viral replicon, or a sequence encoding the same, the replicon being preferably constructed based on a Becurtovirus, or a Curtovirus genome, wherein the at least one viral replicon further comprises: (ii) at least one genome editing system comprising at least one nucleic acid guided nuclease, nickase or an inactivated nuclease, or a sequence encoding the same, and optionally, in case a CRISPR system is used, at least one guide molecule, or a sequence encoding the same; (iii) and, optionally at least one repair template, or a sequence encoding the same; (b) cultivating the cell under conditions allowing the expression and/or assembly of the at least one genome editing system and the at least one guide molecule and/or optionally at least one repair template; and (c) obtaining at least one modified cell; and (d) optionally obtaining at least one plant, plant tissue, organ, or seed regenerated from the at least one modified cell.
In a modification of the above first aspect, there is provided a method forthe targeted modification of at least one genomic target sequence in a plant cell preferably from a plant of the family of Solanaceae, preferably a cell of a plant from the genus Nicotiana, more preferably a cell of a plant of the species Nicotiana tabacum or Nicotiana benthamiana may be provided, wherein the method comprises the following steps: (a) introducing into the cell (i) at least one viral replicon, or a sequence encoding the same, the replicon being preferably constructed based on a Becurtovirus, or a Curtovirus genome, wherein the at least one viral replicon further comprises: (ii) at least one genome editing system comprising at least one nucleic acid guided nuclease, nickase or an inactivated nuclease, or a sequence encoding the same, and optionally, in case a CRISPR system is used, at least one guide molecule, or a sequence encoding the same; (iii) and, optionally at least one repair template, or a sequence encoding the same; (b) cultivating the cell under conditions allowing the expression and/or assembly of the at least one genome editing system and the at least one guide molecule and/or optionally at least one repair template; and (c) obtaining at least one modified cell; and (d) optionally obtaining at least one plant, plant tissue, organ, or seed regenerated from the at least one modified cell.
The present invention hereby solves the problem to provide methods and systems for increasing the efficiency of targeted modification of a nucleotide sequence of interest in the genome of a plant or a plant cell recalcitrant to modifications with state of the art methods and thus provides efficient and robust methods to regenerate new plants from the transformed plant cells, or plant material.
Since the agronomically highly relevant Beta vulgaris plant is not a natural host forthe Mastrevirus BeYDV, in sugar beet BeYDV-based GVRs do not replicate and cannot interact with host factors as efficient as in model plants, e.g., Nicotiana tabacum or Arabidopsis. Therefore, no suitable sugar beet Geminivirus-based replicons can be obtained starting from Geminiviruses per se having a low tropism or infectious and replicative potential in this commercially relevant plant. This was confirmed by quantifying and comparing the replication intensities of GVRs based on BeYDV, BSCTV (Beet severe curly top virus) and BCTIV (Beet curly top iran virus) in sugar beet and also in Nicotians as shown in the below examples, which perfectly illustrates that certain GVRs, and particularly certain BCTIV- and BSCTV-based GVRs, show the important feature that these replicons are suitable for efficient transformations of indeed recalcitrant plant species and, what is more, for plant species of various families.
According to the disclosure provided herein, the limitations of using BeYDV-based replicons in Beta vulgaris, particularly in sugar beet, are overcome by using GVRs, derived from Geminiviruses from different viral genera. The present invention thus specifically provides replicons of two viruses for the heterologous expression of genome editing components in Beta vulgaris, namely:
(i) Beet curly top iran virus (BCTIV, SEQ ID NO: 22), belonging to the genus Becurtovirus; and
(ii) Beet severe curly top virus (BSCTV, SEQ ID NO: 23), belonging to the genus Curtovirus.
Using GVRs derived from BSCTV or BCTIV instead of BeYDV leads to a significantly increased replication intensity in Beta vulgaris leading to high expression levels of the heterologous sequence. This heterologous sequence can for instance encode GE components as well as optionally a repair template and thereby might enhance HDR-mediated genome editing efficiency. Furthermore, GVRs can revert host cells into the S-phase of the cell cycle, which stimulates homology-dependent recombination and thus increases efficiency additionally.
In particular, species like for example Beta vulgaris (such as sugar beet, fodder beet, red beet, or chard) are well known as being very recalcitrant in transgenic modifications. The same applies for other dicotyledonous plants, in particular plants within the taxonomic family of Amaranthacea. After establishing suitable viral replicons for recalcitrant plants, the inventors found that certain of these replicons can have a broad spectrum oftropism for other target plants, e.g., non-crop plants. This indicates, for example, that the use of Geminivirus replicons (GVRs) derived from sugar beet- infecting viruses is not restricted to sugar beet but can be extended to other non-crop species like tobacco ( Nicotiana benthamiana) belonging to the family Solanaceae). To achieve one of the main objects of the present invention to establish suitable viral vectors for recalcitrant plants, it is thus necessary to first establish a new viral replicon very specific for the recalcitrant plant species, as usual viral vectors, including GVRs, will not be efficient. The viral replicons established may additionally be suitable to transform other target plants effectively, particularly those target plants known to be easy or relatively easy to transform or transfect.
In one embodiment, the target plant to be transformed according to the methods of the present invention is thus selected from a plant belonging to the family Amaranthacea or Solanaceae. In preferred embodiments, the methods disclosed herein are thus genotype-independent based on the preceding efforts to provide new GVRs based on specific (Be-)Curtoviruses. A genomic target sequence of interest according to any of the embodiments described herein may be an endogenous or isolated nucleic acid region of a plant cell or organism including any organellar genomic target sequence.
In a preferred embodiment, the at least one viral replicon, or a sequence encoding the same, may be a replicon being constructed based on a Becurtovirus, or a Curtovirus genome. It was surprisingly found that completely new Geminivirus-based replicons (GVRs) derived from BSCTV or BCTIV instead of BeYDV lead to a significantly increased replication intensity in Beta vulgaris and other Amaranthacea plants leading to high expression levels of the heterologous sequence, which for instances encodes GE components as well as optionally a repair template and thereby might enhance HDR-mediated genome editing efficiency. Furthermore, this specific GVRs can revert host cells into the S-phase of the cell cycle, which stimulates homology-dependent recombination and thus increases efficiency additionally. A viral replicon as used herein thus refers to an expression construct based on a specific naturally occurring virus and comprising a DNA molecule or RNA molecule, or a region of DNA or RNA, that can replicate from a single origin of replication, wherein the viral replicon maintains relevant characteristics of the native virus, in particular regarding the plant tropism and its replication efficiency and further beneficial effects, but is modified to be used as expression construct and does not comprise all virulence factors of the native virus.
As introduced above, Beet curly top iran virus (BCTIV) is a new and not yet detailed studied member of Becurtoviruses with a unique nanonucleotide in the LIR and a rather different member of the family Geminiviridae. This virus encodes five genes (V1 , V2 and V3 on the virion-sense strand and Rep and C2 genes on the complementary-sense strand). The genome sequence and structure of the virion-sense stand of Becurtoviruses is similar to Curtoviruses while the complementary-sense strand has similarity to the Mastreviruses. Beet severe curly top virus (BSCTV) and Beet curly top virus (BCTV) are members of Curtoviruses. Curtoviruses are monopartite Geminiviruses that are vectored by leafhoppers, often in a species-specific manner and rather different to Mastreviruses. BCTV encodes usually 7 genes (V1 , V2, V3 on virion-sense strand and Rep, C2, C3 and C4 on the complementary-sense strand) without SIR sequence. Therefore, Becurtovirus and Curtovirus are different in genomic architecture to other Geminivirus replicon systems and thus need certain optimizations to be suitable at all for use as viral replicons, as comparable replicons are not available at date. Obviously, specific adaptations within the genomic structure need to be made to (i) guarantee general functionality of a new viral replicon and (ii) to maintain and improve the replication efficiency of the respective viral replicon, which is easily disturbed when modifying and re-purposing a naturally occurring virus.
According to the findings presented herein, heterologous expression of nucleic acid material for the purpose of GE is achieved using a specifically designed viral replicon derived from, in particular, a Beta vulgaris infecting Geminivirus (GV), or particularly modified variants of said GV. Such modified GV variants have been demonstrated to be suitable for heterologous expression of a nucleotide sequence of interest in planta. Thereby, the artificially created Geminivirus replicons (GVRs) of the present invention replicate efficiently inside host cells and therefore produce high amounts of replicons, in turn producing high expression of genome editing effector molecules cloned into the respective GVR.
Additionally, in certain embodiments, this can help to increase the abundance of donor or repair templates available for HDR, thereby considerably enhancing the targeting efficiency and accuracy.
In addition to rolling circle replication, GVRs as studied and constructed herein, replicate inside host cells also via homologous recombination (HR)-dependent replication, which may revert host plant cells to the S-phase. The S-phase-like endocycle is favorable for HR if, for example, supplemented with site-specific nucleases and complementary donor template.
In one embodiment of the invention, the viral replicon may be provided as single-component replicon for BCTIV, BSCTV or BCTV. For example, a BCTIV replicon may contain LIR, SIR and Rep, or a BCTV or BSCTV replicon may contain LIR and Rep, but no SIR sequence. For the specific replicons newly established and tested, the inventors further found that HDR-based GE efficiency can be significantly increased by using the specific (Be-)Curtovirus GVR, as these viral replicons were constructed in a way maintaining their effect of reverting a plant target cell in the S-phase of the cell cycle, which is highly favorable for HDR to occur, and which stimulates HDR- events.
In one embodiment, a viral replicon according to the present invention may thus be used to revert a host cell of interest into the S-phase and/or to improve HDR-mediated GE efficiency by specifically combining the effects inherent to the new viral replicons of interest as cargo and the GE inserts inserted into these GVR tools. This observation is supported by recent studies done in Arabidopsis thaliana infected with the Geminivirus Euphorbia yellow mosaic virus (EuYMV) (Richter, K. S., et al. (2016). "The recombination mediator RAD51D promotes geminiviral infection." Virology 493: 113-127.).
In a further aspect, there is thus provided a viral replicon optimized for plant transformation and based on the relevant findings on new GVRs based on (Be-)curtovirus genomes as disclosed herein, wherein the viral replicon may be encoded by a sequence according to SEQ ID NO: 4 or 6, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
In one embodiment, BCTIV- and BSCTV-GVRs may thus be individually equipped with various GE system components or tools, which are necessary to mediate SDN-1 events and optionally with at least one repair template to mediate SDN-2/3 kind modifications.
In certain embodiments, introduction of at least one viral replicon may be achieved by biological or physical means, including direct application to a cell, transfection, transformation, including transformation by Agrobacterium spp., preferably by Agrobacterium tumefaciens, biolistic bombardment, transfection using chemical agents, including polyethylene glycol transfection, or any combination thereof. In a preferred embodiment, the viral replicon may be integrated into a vector suitable for Agrobactehum-me0\ate0 plant (cell) transformation such as a binary vector. In another preferred embodiment, the viral replicon may be integrated into a vector suitable for transformation via biolistic bombardment; this is not necessarily a binary vector.
In certain embodiments, the at least one viral replicon, or the sequence encoding the same, may be constructed based on a Beet curly top iran virus Becurtovirus genome, a Spinach Curly Top Arizona Virus Becurtovirus genome, a Beet severe curly top virus Curtovirus genome, a Beet curly top virus Curtovirus genome, a Beet mild curly top virus Curtovirus genome, or a Spinach severe curly top virus Curtovirus genome, preferably a genome selected from SEQ ID NOs: 22, 23, 27 or 28, or a genome from a homologous virus to the respective sequences belonging to the genus Becurtovirus or Curtovirus, wherein a homologous virus may be understood as a naturally occurring plant infecting virus having a comparable genome architecture as a Becurtovirus or Curtovirus and thus belonging to the same genus of Becurtovirus or Curtovirus, but having certain sequence variations.
The at least one viral replicon according to the various aspects and embodiments disclosed herein may be encoded by a sequence according to SEQ ID NO: 4 or 6, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
Whenever the present disclosure relates to the percentage of the homology or identity of nucleic acid or amino acid sequences to each other over the entire length of the sequences to be compared to each other, wherein these identity or homology values define those as obtained by using the EMBOSS Water Pairwise Sequence Alignments (nucleotide) program (www.ebi.ac.uk/Tools/psa/emboss_water/nucleotide.html) for nucleic acid sequences or the EMBOSS Water Pairwise Sequence Alignments (protein) program (www.ebi.ac.uk/Tools/psa/emboss_water/) for amino acid sequences. Those tools provided by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI) for local sequence alignments use a modified Smith-Waterman algorithm (see www.ebi.ac.uk/Tools/psa/ and Smith, T.F. & Waterman, M.S. “Identification of common molecular subsequences” Journal of Molecular Biology, 1981 147 (1 ): 195-197). When conducting an alignment, the default parameters defined by the EMBL-EBI are used. Those parameters are (i) for amino acid sequences: Matrix = BLOSUM62, gap open penalty = 10 and gap extend penalty = 0.5 or (ii) for nucleic acid sequences: Matrix = DNAfull, gap open penalty = 10 and gap extend penalty = 0.5.
Generally, any suitable delivery method to introduce the components (i) or (ii) and optionally (iii) according to step (a) of the methods of the present invention into a cell can be applied, depending on the target cell. The term "introducing" as used herein thus implies a functional transport of a biomolecule or genetic construct (DNA, RNA, single- or double-stranded, protein, comprising natural and/or synthetic components, or a mixture thereof) into a cell or into a cellular compartment of interest, e.g. the nucleus or an organelle, or into the cytoplasm, which allows the transcription and/or translation and/or the catalytic activity and/or binding activity, including the binding of a nucleic acid molecule to another nucleic acid molecule, including DNA or RNA, or the binding of a protein to a target structure within the cell, and/or the catalytic activity of an enzyme such introduced, optionally after transcription and/or translation.
A variety of delivery techniques may be suitable according to the methods of the present invention for introducing the components (i) or (ii) and optionally (iii) into a cell, in particular a plant cell, the delivery methods being known to the skilled person, e.g. by choosing direct delivery techniques ranging from polyethylene glycol (PEG) treatment of protoplasts, procedures like electroporation, microinjection, silicon carbide fiber whisker technology, viral vector mediated approaches and particle bombardment. A common biological means, and a preferred cargo according to the present invention, is transformation with Agrobacterium spp. which has been used for decades for a variety of different plant materials. Viral vector mediated plant transformation represents a further strategy for introducing genetic material into a cell of interest.
In certain embodiments according to the various aspects of the present invention the at least one viral replicon comprising the at least one genome editing system may thus be introduced into the cell by transformation or transfection, preferably by Agrobacterium- mediated transformation, biolistic bombardment, micro- or nanoparticle delivery, or by chemical transfection. Specific vectors have been constructed for use according to the present disclosure and optimized for efficiently delivering the viral replicons and the encoded sequences into a cell of interest.
In one embodiment, there may thus be provided at least one viral replicon comprising at least one genome editing system, wherein the replicon is introduced into the cell by Agrobacterium- mediated transformation, wherein the at least one viral replicon is inserted into a binary vector, preferably wherein the binary vector comprising the at least one viral replicon has a sequence of SEQ ID NOs: 5 or 7 to 15, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
Further vectors, e.g., artificial vectors suitable as shuttle vectors, so-called because they are able to replicate in multiple hosts, e.g., E. coli and Agrobacterium comprising a transfer DNA (T-DNA) binary system necessary for agrobacterium transformation may be used and are available to the skilled person.
In preferred embodiments, the methods for the targeted modification of at least one genomic target sequence in a plant cell are used to genome editing (GE) purposes. The GE strategy may be an SDN-1 strategy exclusively introducing GE tools of interest, but no further foreign nucleic acid material. In other embodiments, SDN-2/3 strategies additionally introducing at least one repair template ordonortemplate, said terms being used interchangeably herein, may be desired. Both strategies have been shown to be compatible with the specific viral replicons designed.
The at least one genome editing system (GE system) according to the present disclosure may preferably comprise at least one nucleic acid guided nuclease, nickase or an inactivated nuclease, or a sequence encoding the same, and at least one guide molecule, or a sequence encoding the same. A "(targeted) modification" of at least one genomic target sequence in the context of the present invention as mediated by a GE system of interest refers to any change of a (nucleic acid) sequence that results in at least one difference in the (nucleic acid) sequence distinguishing it from the original sequence. The at least one "(targeted) modification" can be introduced as a consequence of the endogenous repair mechanism (e.g., as a consequence of NHEJ and other known endogenous repair mechanisms) after the at least one GE system introduced in a targeted way was active, or as a direct consequence of the at least one GE system, e.g., a base editor system, or another fusion system, or as a consequence of a repair template (RT) introduced at the site of a single- or double stranded break as introduced by at least one GE system. The genomic target sequence to be modified in a targeted manner can be present in the nuclear, or in the plastid genome, including, but not being limited thereto, the mitochondrial, and the chloroplast genome. Furtherthe genomic target sequence to be modified may be present in a cell as a target nucleic acid sequence present in a cell extra-chromosomally, for example, as autonomously replicating plasmid, or as nucleic acid plasmid transiently introduced into a cell, but not (fully) integrated into the genome of a target cell. In particular, a modification can be achieved by insertion or addition of one or more nucleotide(s), or substitution or deletion of one or more nucleotide(s) of the original sequence, e.g., a targeted point mutation as mediated by a singlenucleotide exchange, or any combination of these. Specifically, this implies that a targeted modification can comprise more than one modification mediated simultaneously, for example, more than one point mutation, wherein the individual point mutations can be at least one synonymous mutation (resulting in a difference in the nucleic acid sequence, yet not in an exchange of the encoded amino acid sequence, when the sequence encodes an amino acid sequence) together with at least one non-synonymous mutation. Further, a targeted modification can comprise introducing at least one point mutation (synonymous and/or non-synonymous) together with either one of a deletion and/or an insertion in one and the same experiment, i.e., simultaneously. A targeted modification is introduced using site-specific tools such as at least one nucleic acid guided nuclease, or a variant thereof (including a nickase, or a nuclease-dead variant) which recognizes and/or cuts the target at a specific location. If two or more different guide nucleic acid sequences are used, it is possible to target multiple sites in the genomic target region and introduce "multiple modifications". The "multiple modifications" may also be made, for example, in the same gene, or the "multiple modifications" may be made into regions of the genome not directly connected, e.g., modifications in different chromosomes, and the like. More than one, or "multiple modifications" can be introduced, by way of example, by providing a repair template (RT) harboring all desired modifications in the form of at least one point mutation and, optionally, the capacity to mediate an insertion or deletion in addition to the point mutations. To this end, more than one guide RNA or crRNA may be used when "multiple modifications" are of interest, or one one guide RNA or crRNA may be used together with a RT carrying more than one modification to be effected.
A preferred GE system according to the present disclosure is preferably based on a CRISPR system comprising at least one CRISPR nuclease and at least one guide RNA. A "site-directed nuclease" or "site-specific nuclease" can be a single stranded DNA break (SSB) inducing enzyme or a double stranded DNA break (DSB) inducing enzyme, which preferably recognizes a predetermined site in the genome of targeted cell.
A "CRISPR nuclease", as used herein, is a specific form of a site-directed nuclease and refers to any nucleic acid guided nuclease, which has been identified in a naturally occurring CRISPR system, which has subsequently been isolated from its natural context, and which preferably has been modified or combined into a recombinant construct of interest to be suitable as tool for targeted genome engineering. Any CRISPR nuclease can be used and optionally reprogrammed or additionally mutated to be suitable for the various embodiments according to the present invention as long as the original wild-type CRISPR nuclease provides for DNA recognition, i.e., binding properties. CRISPR nucleases also comprise mutants or catalytically active fragments or fusions of a naturally occurring CRISPR effector sequences, or the respective sequences encoding the same. A CRISPR nuclease may in particular also refer to a CRISPR nickase or even a nuclease-dead variant of a CRISPR polypeptide having endonucleolytic function in its natural environment. The CRISPR nucleases include CRISPR/Cas systems, including CRISPR/Cas9 systems, CRISPR/Cpfl systems, CRISPR/C2C2 systems, CRISPR/CasX systems, CRISPR/CasY systems, CRISPR/Cmr systems, CRISPR/MAD systems, including, for example, CRISPR/MAD7 systems and CRISPR/MAD2 systems, CRISPR/CasZ systems and/or any combination, variant, or catalytically active fragment thereof. A nuclease may be a DNAse and/or an RNAse, in particular taking into consideration that certain CRISPR effector nucleases have RNA cleavage activity alone, or in addition to the DNA cleavage activity.
A "CRISPR system" is thus to be understood as a combination of a CRISPR nuclease or CRISPR effector, or a nickase or a nuclease-dead variant of said nuclease, or a functional active fragment or variant thereof together with the cognate guide RNA (or pegRNA orcrRNA) guiding the relevant CRISPR nuclease.
A nucleic acid guided nuclease, or site-specific or site-directed nuclease, of any kind as delivered with a viral replicon according to the present invention thus recognizes a certain protospacer adjacent motif (PAM) at the target site, which is required to be present for the nuclease to cut the target site. The "PAM specificity" of a nuclease defines, which PAM(s) the nuclease recognizes. For example, certain variations of a PAM or different PAMs may result in cleavage. The different variants or different PAMs may provide a varying degree of nuclease activity at a target site. In the context of the present invention, a nuclease is considered to “recognize” a certain PAM, when the indel percentage at a certain site with the PAM, normalized by transformation efficiency in the system used, is at least 10%, preferably at least 20%, at least 30%, at least 40%, at least 50% or at least 60%. In this context, a “higher activity” of a nuclease refers to a higher indel percentage at a certain target site compared to another nuclease. An “indel” refers to an insertion or a deletion of one or more nucleotides at the target site, which is due to site specific nuclease activity at the target site. The frequency with which indels occur at the target site can be used as a measure for site specific nuclease activity. ln preferred embodiments, the at least one nucleic acid guided nuclease, nickase or an inactivated nuclease, or a sequence encoding the same, of the genome editing system may thus be selected from the group consisting of a CRISPR/Cas system, preferably from a CRISPR/Cfp1 system, a CRISPR/MAD7 system, a CRISPR/MAD2 system, a CRISPR/Cas9 system, a CRISPR/CasX system, a CRISPR/CasY system, or a CRISPR/Csm system, or any combination, variant, or catalytically active fragment thereof.
In certain embodiments, the GE system may be a base editor (BE) system. Base editors, including CBEs (base editors mediating C to T conversion) and ABEs (adenine base editors mediating A to G conversion), are powerful tools to introduce direct and programmable mutations without the need for double-stranded cleavage (Komor ef a/., Nature, 2016, 533(7603), 420-424; Gaudelli et al., Nature, 2017, 551 , 464-471). In general, base editors are composed of at least one DNA targeting module and a catalytic domain that deaminates cytidine or adenine. All four transitions of DNA (A®T to G®C and C®G to T®A) are possible as long as the base editors can be guided to the target site. Originally developed for application in mammalian cell systems, both BEs and ABEs have been optimized and applied in plant cell systems. Efficient base editing has been shown in multiple plant species ( Zong et al ., Nature Biotechnology, vol. 25, no. 5, 2017, 438-440; Yan etal., Molecular Plant, vol. 11, 4, 2018, 631-634; Hua et at, Molecular Plant, vol. 11, 4, 2018, 627-630). Base editors have been used to introduce specific, directed substitutions in genomic sequences with known or predicted phenotypic effects in plants and animals. But they have not been used for directed mutagenesis targeting multiple sites within a genetic locus or several loci to identify novel or optimized traits.
Base editing as detailed above, does not cut the double-stranded DNA but instead uses the CRISPR targeting machinery to shuttle an additional enzyme to a desired sequence, where it converts a single nucleotide into another. Many genetic traits in plants and certain susceptibility to diseases caused by plant pathogens are caused by a single nucleotide change, so base editing offers a powerful alternative for GE. But the method has intrinsic limitations and is said to introduce off-target mutations, which are generally not desired for high precision GE.
In yet another embodiment, the GE system may be a Prime Editing system ( Anzalone et al. (2019). Search-and-replace genome editing without double-strand breaks (DSBs) or donor DNA. Nature, 1-1.). In contrast to Base Editing, Prime Editing (PE) systems steer around the shortcomings of earlier CRISPR based GE techniques by heavily modifying the Cas9 protein and the guide RNA. The altered Cas9 only "nicks" a single strand of the double helix, instead of cutting both. The new guide RNA, called a pegRNA (prime editing extended guide RNA), contains an RNA template for a new DNA sequence, to be added to the genome at the target location. That requires a second protein, attached to Cas9 or a different CRISPR effector nuclease: a reverse transcriptase enzyme, which can make a new DNA strand from the RNA template and insert it at the nicked site. To this end, an additional level of specificity is introduced into the GE system in view of the fact that a further step of target specific nucleic acid::nucleic acid hybridization is required. This may significantly reduce off-target effects. Further, the PE system may significantly increase the targeting range of a respective GE system in view of the fact that BEs cannot cover all intended nucleotide transitions/mutations (C®A, C®G, G®C, G®T, A®C, A®T, T®A, and T®G) due to the very nature of the respective systems, and the transitions as supported by BEs may require DSBs in many cell types and organisms.
In certain embodiments according to the various aspects of the present invention, the at least one GE system thus may further comprise at least one PE system and plant host compatible reverse transcriptase, preferably a modified reverse transcriptase, wherein the reverse transcriptase is preferably used in combination with a nucleic acid guided nickase, and/or at least one cytidine or adenine deaminase, preferably wherein the at least one cytidine or adenine deaminase is independently selected from an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase, preferably a rat-derived APOBEC, an activation-induced cytidine deaminase (AID), an ACF1/ASE deaminase, an ADAT family deaminase, an ADAR2 deaminase, or a PmCDAI deaminase, a TadA derived deaminase, and/or a transposon, or a sequence encoding the aforementioned at least one enzyme, or any combination, variant, or catalytically active fragment thereof.
Any nucleic acid sequence of the GE systems disclosed herein may be “codon optimized” for the codon usage of a plant target cell of interest. This means that the sequence is adapted to the preferred codon usage in the organism that it is to be expressed in, i.e. a “target cell of interest”. If a nucleic acid sequence is expressed in a heterologous system, codon optimization increases the translation efficiency significantly.
A GE system, in particular a CRISPR system, comprises at least one nucleic acid guided nuclease and at least one "guide molecule" or "guide nucleic acid sequence" (usually called and abbreviated as guide RNA, crRNA, crRNA+tracrRNA, gRNA, sgRNA), which recognizes a target sequence to be cut by the nuclease. The at least one "guide nucleic acid sequence" or "guide molecule" comprises a "scaffold region" and a "target region". The "scaffold region" is a sequence, to which the nucleic acid guided nuclease binds to form a targetable nuclease complex. The scaffold region may comprise direct repeats, which are recognized and processed by the nucleic acid guided nuclease to provide mature crRNA. A pegRNA may comprise a further region within the guide molecule, the so-called "primer-binding site". The "target region" defines the complementarity to the target site, which is intended to be cleaved. A crRNA as used herein may thus be used interchangeably herein with the term guide RNA in case it unifies the effects of meanwhile well-established CRISPR nuclease guide RNA functionalities. Certain CRISPR nucleases, e.g., Cas9, may be used by providing two individual guide nucleic acid sequences in the form of a tracrRNA and a crRNA, which may be provided separately, or linked via covalent or non-covalent bonds/interactions. The guide RNA may also be a pegRNA of a Prime Editing system as further disclosed above.
A "target region/sequence" defines the complementarity to the target site, which is intended to be cleaved, and a "genomic target region/sequence" is a region in the genome, including the organellar genome, of the target cell, which is to be modified using the GE system with a viral replicon of the present invention. The target region can be an endogenous sequence, e.g. an endogenous target gene, or an isolated nucleic acid region, which is not part of the genome of the target cell but e.g. present on a plasmid or an artificial chromosome.
A "repair template" (RT) or "donor template/nucleic acid" represents a single-stranded or double- stranded nucleic acid sequence, which can be provided during any genome editing causing a double-strand or single-strand DNA break to assist the targeted repair of said DNA break by providing a RT as template of known sequence assisting homology-directed repair. The RT may comprise "symmetric or asymmetric homology arms", which provide homology to the sequences flanking the double-strand break introduced by the nuclease and thus promote error-free homology directed repair. The repair template may also comprise at least one chemically modified base or backbone, wherein a "chemically modified base" is present in the repair template, when at least one nucleobase has been modified to carry one or more substituent(s) or label(s) or one or more nucleotide(s) carry a molecule other than a nucleobase instead of a nucleobase. A "chemically modified back bone" is present in the repair template, for example, when the phosphate back bone carries at least one modification such as e.g. a phosphorothioate bond, or, for example, when at least one nucleobase has been modified to carry one or more substituent(s) or label(s) or one or more nucleotide(s) carry a molecule other than a nucleobase instead of a nucleobase. A repair template can thus be provided during any genome editing causing a doublestrand and/or single-strand DNA break to assist the targeted repair of said DNA break by providing a RT as template of known sequence assisting homology-directed repair. For example, a repair template can be provided as individual molecule, or a sequence encoding the same, or it may be associated with a guide RNA of a CRISPR nuclease, wherein the association can be of non- covalent nature (complementary base pairing), or the molecules can be physically attached to each other by a covalent bond. The latter embodiment may increase the availability of the at least one repair template of interest at the target site in a genome, where a targeted double- or single- stranded break was introduced which is to be repaired.
In certain embodiments, the at least one repair template may comprise symmetric or asymmetric homology arms and/or the at least one repair template may comprise at least one chemically modified base and/or backbone.
In certain embodiments, it may be preferably to add a repair template to the GE system to obtain a GE event in a highly targeted way for SDN-2/3 settings. Therefore, the at least one genome editing system may comprise at least one repair template, and wherein the at least one repair template may comprise or encode a double- and/or single-stranded nucleic acid sequence, and/or wherein the at least one repair template may be part of the at least one guide molecule.
In another aspect according to the present invention, what is provided may be a plant, plant cell, tissue, organ, or seed obtainable by or obtained by a method according to any of the methods for the targeted modification of at least one genomic target sequence in a plant cell as disclosed herein. As obtaining at least one plant, plant tissue, organ, or seed by regenerating the same from at least one cell modified herein is readily available to the skilled person in the field of plant biotechnology and breeding, the plant material modified in a targeted manner can be used to obtain a whole plant organism, or a part thereof, carrying the desired modification, preferably in an inheritable manner. "Regenerating / regeneration" in this context is to be understood broadly and refers to any kind of in plants, in vitro, or a combined in vivo/ex vivo cultivation of at least one modified plant cell, tissue, organ, or material.
Suitable conditions to cultivate plant cells and plant material as modified according to the methods of the present invention in vitro and in vivo are disclosed herein and are known to the skilled person.
To avoid negative effects on the callus development and/or shoot regeneration, which steps may in certain embodiments be desired for a regeneration step, an inducible promoter system may be used for the expression of the viral replication initiator protein (Rep). Such inducible systems include but are not limited to inducible promoter systems such as the abscisic acid-inducible promoter (SEQ ID NOs: 1 and 2) or a glucocorticoid receptor-based system inducible by dexamethasone (Figure 2, SEQ ID NO: 3). Further inducible systems compatible for use in plant cells are known to the skilled person.
In certain embodiments, the methods of the present invention may be performed by transiently introducing at least one viral replicon, or a sequence encoding the same and/or at least one genome editing system comprising at least one nucleic acid guided nuclease, nickase or an inactivated nuclease, or a sequence encoding the same, and at least one guide molecule, or a sequence encoding the same and/or and, optionally at least one repair template, or a sequence encoding the same, whereas in other embodiments, the tools may be introduced in a stable way. A combination of transient and stable transformations is possible in view of the fact that at least two components are inserted into the at least one cell to be modified. "Transient" implies that the tools are only temporarily expressed and afterwards degraded by the cell, whereas "stable" implies that at least one of the tools is integrated into the nuclear and/or organellar genome of the cell to be modified.
In one aspect, an expression construct is provided, i.e., a nucleic acid cassette comprising several elements fortranscribing and/or translating encoded elements in at least one target cell of interest, which may comprise or encode: (i) at least one viral replicon based on a (Be-)Curtovirus as disclosed according to the various aspects and embodiments herein, and (ii) a vector, preferably a (binary) vector as defined above suitable for Agrobacterium- mediated transformation, and (iii) at least one component of at least one a genome editing system, wherein the genome editing system comprises or encodes: (a) at least one nucleic acid guided or site- specific nuclease, nickase or an inactivated nuclease, or at least one site-directed nuclease, nickase or an inactivated nuclease, and optionally at least one nuclear or organellar localization sequence; and (b) at least one guide molecule for guiding the at least one nucleic acid guided nuclease, nickase or an inactivated nuclease and optionally for activating at least one reverse transcriptase; and (c) optionally at least one repair template; and (d) optionally at least one reverse transcriptase; and (iv) wherein the expression construct comprises or encodes at least one regulatory sequence, wherein the regulatory sequence is selected from the group consisting of a core promoter sequence, a proximal promoter sequence, a cis regulatory sequence, a trans regulatory sequence, a locus control sequence, an insulator sequence, a silencer sequence, an enhancer sequence, a terminator sequence, an intron sequence, and/or any combination thereof.
In one embodiment of the expression construct described above, the construct comprises or encodes at least one regulatory sequence, wherein the regulatory sequence is selected from the group consisting of a core promoter sequence, a proximal promoter sequence, a cis regulatory sequence, a trans regulatory sequence, a locus control sequence, an insulator sequence, a silencer sequence, an enhancer sequence, a terminator sequence, an intron sequence, and/or any combination thereof.
In another embodiment of the expression construct described above, the regulatory sequence comprises or encodes at least one promoter selected from the group consisting of ZmUbH , BdUbHO, ZmEfl , a double 35S promoter, a rice U6 (OsU6) promoter, a rice actin promoter, a maize U6 promoter, PcUbi4, Nos promoter, AtUbHO, BdEF1 , MeEF1 , HSP70, EsEF1 , MdHMGRI , or a combination thereof.
In a further embodiment of the expression construct described above, the at least one intron is selected from the group consisting of a ZmUbM intron, an FL intron, a BdUbHO intron, a ZmEfl intron, a AdH1 intron, a BdEF1 intron, a MeEF1 intron, an EsEF1 intron, and a HSP70 intron.
In one embodiment of the expression construct according to any of the embodiments described above, the construct comprises or encodes a combination of a ZmUbil promoter and a ZmUbil intron, a ZmUbil promoter and FL intron, a BdUbMO promoter and a BdUbMO intron, a ZmEfl promoter and a ZmEfl intron, a double 35S promoter and a AdH1 intron, or a double 35S promoter and a ZmUbil intron, a BdEF1 promoter and BdEF1 intron, a MeEF1 promoter and a MeEF1 intron, a HSP70 promoter and a HSP70 intron, or of an EsEF1 promoter and an EsEF1 intron.
In addition, the expression construct may comprise at least one terminator, which mediates transcriptional termination at the end of the expression construct or the components thereof and release of the transcript from the transcriptional complex.
In one embodiment of the expression construct according to any of the embodiments described above, the regulatory sequence may comprise or encode at least one terminator selected from the group consisting of nosT, a double 35S terminator, a ZmEfl terminator, an AtSac66 terminator, an octopine synthase (ocs) terminator, or a pAG7 terminator, or a combination thereof. A variety of further suitable promoter and/or terminator sequences for use in expression constructs for different plant cells are well known to the skilled person in the relevant field.
A "nuclear/organellar localization signal/sequence" (abbreviated as NLS/OLS, respectively) refers to an amino acid sequence, or a sequence encoding the same, which is usually added at the C- terminus and/or the N-terminus of a polypeptide or protein, or the sequence encoding the same, which causes the polypeptide or protein to be imported into the cellular nucleus or organelle by nuclear transport or intracellular trafficking in a host cell of interest. More than one NLS or OLS sequence may be present in a target protein, usually a GE system effector nuclease, nickase or nuclease-dead variant thereof. Suitable NLS and OLS sequences for different plant cells are described in the literature.
In one embodiment, the expression construct may additionally comprise or encode at least one self-cleaving ribozyme, preferably at least one hammerhead ribozyme and/or at least one hepatitis-delta virus (HDV) ribozyme and/or at least one artichoke, sunflower or rice HDV-like ribozyme.
A "self-cleaving ribozyme" is an RNA molecule that is capable to catalyse its own cleavage at a specific site. Upon transcription, self-cleaving ribozymes fold into a specific structure, sometimes requiring the presence of certain metal cations, which induces cleavage of the phosphodiester backbone at a certain position. A number of ribozymes are known, which can be used in a variety of settings. These ribozymes may be particularly suitable according to the present disclosure to "activate" encoded GE system components, e.g., various guide RNAs etc., by cleaving and thus activating these crRNAs/pegRNAs/guide RNAs to make them available for interaction with a CRISPR effector protein of interest which is turn targeted to a target site in the plant genome to be modified in a site-directed way.
The expression construct may thus also comprise at least one ribozyme, which upon transcription cleaves the (RNA) transcript at one or more predetermined location(s). If placed strategically, the ribozyme(s) release and activate the components of the expression construct from the transcript. For example, the sequence encoding the CRISPR nuclease (or variant thereof) may be flanked by a ribozyme at the 5'- and at the 3'-end. The guide nucleic acid sequence(s) may be included between ribozyme and nuclease sequence and can be processed by the certain CRISPR nucleases (e.g., Cpf1 variants and MAD7-type nucleases having an intrinsic RNA-cleavage activity in addition to the DNA-cleavage activity) to provide mature crRNA/pegRNA/guide RNA in turn needed for guidance of the CRISPR effector nuclease or variant thereof.
In yet another embodiment, the expression construct may additionally comprise or encode a sequence for an RNA polymerase II (Pol II) promoter. RNA polymerase II promoters, which constitute the majority of the characterized promoters, cannot be directly used for CRISPR guide RNA production in vivo because of: (1) The primary transcripts of RNA polymerase II promoters undergo extensive processing such as 5'-end capping, 3'-end polyadenylation, and splicing out of the introns. Some of the modifications may thus render the designed guide RNA non-functional; and (2) the mature RNA molecules are transported into cytosol, thus they are physically separated from the intended targets that are located in the nucleus (Zhang et al., doi:10.21769/BioProtoc.2148). That may be the reason why production of guide RNA /n vivo using U6 and U3 snRNA promoters has long been the dominant method (Gao and Zhao, Selfprocessing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J. Integr. Plant Biol. 2014; 56(4):343-349).
As it is known for certain CRISPR systems and effectors, e.g. the Cpf1 system, Cpf1 and orthologs thereof, including LBCpfl and AsCpfl have RNase activities that can excise multiple CRISPR RNAs (crRNAs) from a single Pol ll-driven RNA transcript (Zhong et al., 2017, supra). Therefore, crRNA excised from a Pol ll-driven mRNA transcript may be used in the certain embodiments in the replicons and/or expression constructs and/or binary vectors disclosed herein to effectively provide said crRNAs as guiding RNAs to the cognate CRISPR effector.
According to certain embodiments, a construct or replicon according to the present invention may either comprise at least one self-cleaving ribozyme encoding sequence and at least one RNA pol II promoter, or it may comprise both elements.
According to certain embodiments, a DNA-dependent RNA polymerase type II promoter may thus be used, preferably together with at least one sequence encoding at least one self-cleaving ribozyme to further enhance the activation of the GE system components as encoded by a viral replicon and/or an expression construct according to the present invention. Suitable promoters are available in the prior art and can be combined as replicon and/or expression construct element based on the disclosure provided herein.
Exemplary viral replicons, expression constructs and vectors combining the above elements in various forms to be suitable for the methods disclosed herein are disclosed in the Examples section and are exemplified in the sequence listing. As it will be apparent to the skilled person, certain elements of the replicons, expression constructs and/or vectors are readily exchangeable and can (or must) be adapted for use in a plant cell of interest, whereas in particular, the overall structure of the (Be-)Curtovirus replicon is relevant to obtain certain effects as disclosed herein.
The terms "plant" or "plant cell" as used herein refer to a plant organism, a plant organ, differentiated and undifferentiated plant tissues, plant cells, seeds, and derivatives and progeny thereof. Plant cells include without limitation, for example, cells from seeds, from mature and immature embryos, meristematic tissues, seedlings, callus tissues in different differentiation states, leaves, flowers, roots, shoots, male or female gametophytes, sporophytes, pollen, pollen tubes and microspores, protoplasts, macroalgae and microalgae. The different eukaryotic cells, for example, animal cells, fungal cells or plant cells, can have any degree of ploidity, i.e. they may either be haploid, diploid, tetraploid, hexaploid or polyploid.
The family of Amaranthaceae the preferred plants or plant cells according to the present disclosure belong to comprises a number of relevant food and crop plants. For example, the family comprises the genus Beta, comprising Beta vulgaris ssp. vulgaris var. vulgaris (chard), Beta vulgaris ssp. vulgaris var. conditiva (beetroot / red beet), Beta vulgaris ssp. vulgaris var. crassa/alba (fodder beet) and Beta vulgaris subsp. vulgaris var. altissima (sugar beet in a narrower sense), the genus Amaranthus, the genus Chenopodium, including C. quinoa, or the genus Spinacia including Spinacia oleracea. In certain embodiments, the viral replicons identified herein may be optimized for use in a usually recalcitrant plant of the family Amaranthacea, but the replicons may also be suitable for transforming less or not recalcitrant plants as illustrated with the enclosed Examples.
In yet a further aspect, there is provided a use of a viral replicon as disclosed herein based on a (Be-)Curtovirus, or a use of an expression construct as disclosed herein in various aspects and embodiments, for stimulating homology-directed repair by reverting a host plant cell into the S- phase of the cell cycle, and/or for increasing homology-directed repair-based genome editing.
This aspect is highly desirable in classical plant breeding as well as plant biotechnology. Thereby genetic variation in a specific genomic region, or in a specific nucleotide sequence of interest may be increased, a complete allele or a part like a domain, or an exonic region of a gene of interest may be replaced, or the elimination of undesired genetic components/material closely linked a nucleotide sequence of interest in the genome of a plant (so-called linkage drag), which cannot be easily segregated out by conventional breeding, can be obtained.
The present invention is further described with reference to the following non-limiting Examples.
Examples
Example 1 : Selection of sugar beet infecting Geminivirus strain for replicon construction
Full length clones (FLC) of four SB-infecting Geminivirus strains (BSCTV (Beet severe curly top virus), BCTV (Beet Curly Top Virus), BMCTV (Beet Mild Curly Top Virus), BCTIV (Beet curly top iran virus)) of different genera have been used for Agrobacterium- mediated transformation of sugar beet seedlings to compare viral replication intensity in plants. This initial step was necessary to identify new viruses suitable at all for subsequent viral replicon design in view of the fact that viral replicons for certain dicot target plants like sugar beet and spinach are not readily available and cannot be simply derived from exiting viral replicons having specificity for other target plants.
Highest replication intensity was demonstrated for BSCTV, medium replication level was measured for BCTV and BCTIV and relatively low replication level were shown for BMCTV (Figure 1 A). Based on the experimental results, BSCTV and BCTIV were selected for GVR construction. BSCTV and BCTIV belong to different viral genera than BeYDV.
The design for the GVR construct of BSCTV and BCTIV was adapted and optimized to produce functional replicon constructs in an iterative manner. Additionally, it was demonstrated that not only the DNA levels of the FLC but also the viral DNA levels of the replicons are increased when using the BSCTV-based and the BCTIV-based replicons in comparison to the BeYDV-based replicon (Figure 1 B).
Different Geminivirus genera differ in their genomic structure and gene composition. Therefore, for each viral genus, specific adaptations within the genomic structure need to be made to (i) guarantee general functionality of the GVRs and (ii) further improve the replication efficiency of the respective GVR.
Example 2: Inducible expression of the viral replication initiator protein (Rep) for improved callus development and/or shoot regeneration
To avoid negative effects on the callus development and/or shoot regeneration in Beta vulgaris, preferably in sugar beet, an inducible promoter system may be used forthe expression of the viral replication initiator protein (Rep). Such inducible systems include but are not limited to inducible promoter systems such as the abscisic acid-inducible promoter (SEQ ID NOs: 1 and 2) or a glucocorticoid receptor-based system inducible by dexamethasone (Figure 2, SEQ ID NO: 3).
Example 3: Replicon design
For viral replicon construction, the BCTIV full length clone (FLC, SEQ ID NO: 22) was used as starting material. Using the BCTIV FLC as a template, a PCR-based technique was applied to amplify the viral genome without a 1 ,043 bp fragment which covers the viral ORFs for proteins V1 , V2 and V3. Thereby a viral replicon is created lacking ORFs V1 , V2 and V3. A multi(ple) cloning site (MCS) was introduced into the BCTIV replicon. The final BCTIV replicon thus constructed (SEQ ID NO: 4) contains a repeat of long intergenic region (LIR), small intergenic region (SIR) and C1 (Rep) and C2 (TrAP) genes, which play a role in the virus replication and DNA accumulation.
To allow Agrobacterium- mediated delivery of BCTIV-GVR into plants (which represents one option for introducing the viral replicon efficiently), the replicon was introduced into the T-DNA of binary vector pZFNnptll via Ncol/Pmel sites (see Figure 3, SEQ ID NO: 5).
Further introduction strategies are possible for the developed viral replicons, depending on the experiment and the target material to be treated of interest.
To have further candidates for viral replicon construction, the BSCTV full length clone (SEQ ID NO: 23) was used as further starting material. Using the BSCTV FLC as a template, a PCR-based technique was applied to amplify the viral genome without a 1 ,002 bp fragment which covers the viral ORFs for proteins R2 and R3 and parts of ORF R1 and R4. Thereby a viral replicon is created lacking R2 and R3 and parts of R1 and R4. A multi(ple) cloning site (MCS) was introduced into the BSCTV replicon. The final BSCTV replicon (SEQ ID NO: 6) contains a repeat of LIR and all complementary sense genes including C1, C2, C3 and C4. To allow Agrobacterium- mediated delivery of BSCTV-GVR into plants, the replicon was introduced into the T-DNA of binary vector pZFNnptll via Ncol/Pmel sites (see Figure 4, SEQ ID NO: 7).
Example 4: Introduction of heterologous sequences (e.g. nucleotide sequence of interest or repair template) into the replicons
An expression cassette for GVR-mediated transgene expression (for example 35S::GFP) can be introduced via different cloning sites (for example Spel-sites) present in the MCS of pZFNnptll- BCTIV-Rep/MCS and pZFNnptll-BSCTV-Rep/MCS, respectively (SEQ ID NO: 8 and SEQ ID NO: 9). Genome Editing (GE) components (LbCpfl (SEQ ID NO: 24), crRNA (SEQ ID NOs: 16 to 20), and repair templates (ALS as example, SEQ ID NO: 21)) were introduced into pZFNnptll-BCTIV- Rep/MCS (using SEQ ID NO: 5, producing SEQ ID NOs: 12 and 13) and pZFNnptll-BSCTV- Rep/MCS (using SEQ ID NO: 7, producing SEQ ID NOs: 14 and 15) using the following cloning strategy: first, a pcUbi4::LbCpf1 expression cassette was introduced via XmaUApal sites of the MCS into pZFNnptll-BCTIV-Rep/MCS and via Xma\/Hind\\\ into pZFNnptll-BSCTV-Rep/MCS. In a next step the d35S::crRNA expression cassette was introduced via Sfi\ sites which are present in the MCS of the viral replicons. Finally, the repair template is inserted using a unique Hind\\\ site. When pZFNnptll-BCTIV-Rep/MCS is used for inserting the repair template the Hind\\\ site is located outside of the 3' end of the Tpea terminator, which is part of the pcUbi4::LbCpf1 expression cassette. Therefore, the Hind\\\ site only becomes available after pcUbi4::LbCpf1 expression cassette has been introduced into the viral replicons.
Example 5: Binary vector pZFNnptll, GE components (LbCpfl and crRNAs) and Agrobacterium tumefaciens strain
The viral replicons are embedded in a binary vector named pZFNnptll. This binary vector includes a nptll resistance cassette for in planta selection. To mediate GE in a sugar beet, the target gene, the PcUbi4::Cpf1 expression cassette and a crRNA, which targets a target region within the target gene, were introduced into the viral replicons. In a first version (SEQ ID NOs: 13 and 15, Figure 5 A) the crRNA is embedded in a Pol ll-driven RNA transcript as described before in Zhong et al., 2017. This transcript includes an mRNA encoding the fluorescent protein tDT (Figure 5 A).
The second crRNA version (as included in SEQ ID NOs: 12 and 14) produces the crRNA from a Pol II promoter using the ribozyme-based strategy (He et al., 2017). Thereby, the crRNA production unit is composed of three parts: the 5'-end encoding the Hammerhead (HH) ribozyme, the middle part encoding the crRNA, and the 3'-end encoding the Artichoke HDV-like ribozyme (Figure 5 B). The primary transcripts will undergo self-cleavage catalyzed by the intrinsic nuclease activities of the ribozymes to release the mature and desired crRNA.
The binary vectors were transformed into agrobacterium strain AGL1-1. Notably, other agrobacterium strains available may be used as well.
Example 6: GFP expression
Next, the constructs as established (cf. Examples 4 and 5) were subjected to several rounds of tests. First, green fluorescent protein (GFP) expression levels (transcript level) were compared between a BCTIV-based replicon (SEQ ID NO: 8), a BSCTV-based replicon (SEQ ID NO: 9), a BeYDV-based replicon (SEQ ID NO: 10) and a standard T-DNA vector without viral replicon elements (SEQ ID NO: 11). Therefore, an expression cassette of 1 ,843 bp in size, which expresses the GFP under the 35S promoter, was cloned into GVRs derived from BSCTV, BCTIV and BeYDV. The binary vector pZFNnptll, which includes the 35S::GFP expression cassette, was included as a non-GVR control. The constructs were infiltrated into cotyledons of sugar beet seedlings or tobacco leaves and GFP transcript levels were measured using qRT-PCR (reference gene for sugar beet: GAPDH) after four and six days of infiltration in sugar beet (Figure 6) and after six days of infiltration in tobacco (Figure 7).
In sugar beet, the viral replicons derived from BSCTV and BCTIV clearly outperform BeYDV-GVR and the binary vector control in term of heterologous gene expression. GFP transcript level are strongly increased when expressed by BSCTV or BCTIV-based GVRs compared to the binary vector control and BeYDV-GVR (Figure 6). ln tobacco, BCTIV-GVR and BSCTV-GVR express the GFP gene at a significantly higher levels compared to the binary vector control and BeYDV-GVR at 6 dpi (Figure 7).
This indicates that the use of GVRs derived from sugar beet-infecting viruses is not restricted to sugar beet but can be extended to other non-crop species like tobacco.
Example 7: Expression of GE components (SDN-1)
First, a nucleic acid guided site-specific nuclease (SSN) and a cognate crRNA (SDN-1 technique not relying on adding exogenous nucleic acid material in the form of a repair template) was further tested based on the specific viral replicons tested.
Therefore, a PcUbi4::Cpf1 expression cassette of ~5.2 kb in size and/or a d35S::crRNA expression cassette of 1 .1-1 .7 kb in size, which targets one of five different target regions within the target gene acetolactate synthase (BvALS, SEQ ID NOs: 16 to 20) were introduced into the BCTIV-replicon and the BSCTV-replicon (Figure 5) to mediate genome editing in a sugar beet target gene. For each of the five protospacer two different crRNA versions were tested (tDTcrRNA_ALS1 - ALS5 (e. g. SEQ ID NOs: 13 and 15) or RibocrRNA_ALS1 - ALS5 (SEQ ID NOs: 12 and 14)). The GVRs harboring either only the LbCpfl expression cassette or both, the LbCpfl- and the crRNA-cassette have been infiltrated into cotyledons of sugar beet seedlings using agrobacteria. As non-GVR controls, the corresponding binary vectors without the replicon have been included. Transcript and DNA levels of LbCpfl were quantified by qRT-PCR and qPCR at three different time points post inoculation.
Agrobacterium- mediated infiltration of sugar beet (SB) seedlings with BCTIV-derived GVRs revealed that in comparison to the binary vector controls, LbCpfl transcription (Figure 8 shows RT-PCR results) and DNA levels (Figure 9 shows qPCR results) are strongly increased when expression is mediated by BCTIV-GVR. However, increasing size of the inserted heterologous sequence seems to negatively affect replication ability and/or transcription level of the BCTIV- GVR. The BCTIV-GVR including only the pcUbi4:: LbCpfl expression cassette of ~5.2 kb in size shows a higher transcript level than the BCTIV-GVRs including both the LbCpfl and the crRNA expression cassettes.
Agrobacterium- mediated infiltration of SB seedlings with BSCTV-derived GVRs revealed that LbCpfl transcript and DNA levels are dramatically increased compared to the binary vector controls (Figure 9, see also Figures 11 and 12). For BSCTV-GVR, increasing size of the heterologous insertions seems to not impair replicon function. BSCTV-GVRs including both the LbCpfl and the crRNA expression cassettes show as high transcript levels as the BSCTV-GVR only harboring LbCpfl .
Next, SDN-1 (indel) efficiencies were determined. Cotyledons of sugar beet seedlings were infiltrated with different GVR constructs (derived from BSCTV and BCTIV) containing either only the LbCpfl expression cassette (= negative control for SDN-1 events, since no crRNA is available for target recognition) or both the LbCpfl and the crRNA cassette enabling the quantification of SDN-1. For each GVR construct the respective binary vector without replicon was included as a control. Infiltrated SB leaf explants were harvested 13 (BSCTV-based GVR, Figure 13A) or 12 (BCTIV-based GVR, Figure 13B) days after infiltration (dpi). DNA was extracted and SDN-1 (indel) efficiencies were determined via ddPCR (droplet digital PCR).
Example 8: Expression of GE components (SDN-2/3)
Next, SSN + crRNA + repair template (SDN-2/3) approaches can be tested with the above viral replicons and binary vector systems established. HDR-based genome editing efficiencies in sugar beet are increased by GVR-mediated delivery of (i) site-specific nucleases (SSNs) from the CRISPR/Cas type, (ii) sgRNA and (iii) a repair template (e. g. SEQ ID NO: 21 for BvALS,
Figure 10).
To further improve the efficiency of HDR-mediated GE, the GVR is equipped with a repair template, which will be integrated into the genomic target region during homologous recombination. Since GVRs replicate efficiently inside host cells they produce high amounts of replicons, in turn producing high expression of SSNs and increasing the abundance of donor or repair templates available for HDR, thereby considerably enhancing the targeting efficiency. Therefore, BSCTV- and BCTIV-GVR constructs are equipped with the HDR-based GE components LbCpfl , the crRNA and the repair template. The constructs are used for agrobacterium-mediated infiltration of sugar beet leaf explants.
Leaf samples are taken 10 days post infiltration (dpi) and they are used for DNA extraction and subsequent NGS analysis to quantify efficiencies of HDR events.
Example 9: GVR-based vector system for HDR-based GE in tobacco
Efficiency of GVR-based GE was tested in the transgenic tobacco line Nicotians benthamiana 16c (Philips et al., 2017), which expresses an optimized Aequorea victoria green fluorescent protein (mGFP5-ER, codon optimized and equipped with additional regulatory sequences for the expression in plants (Philips et al., 2017)) constitutively. A single amino acid exchange (T224Y) in GFP causes an altered light emission spectrum that turns GFP to YFP (yellow fluorescent protein (Wachter et al. 1998; Figure 14, SEQ ID NO: 29 and SEQ ID NO: 30)).
The repair template that enables the GFP to YFP mutation consists of two homologous arms, which are complementary to the target region in mGFP5-ER. Two nucleotides within the homologous arms were adjusted, to remove Bsal and Bbs\ restriction sites (Figure 15).
The “core” of the RT (Figure 15, lower panel) includes 23 synonymous mutations, which avoid binding and cutting of the CRISPR complex after RT integration. In addition, the “core” includes 3 non-synonymous mutations (dark grey), which lead to the desired T224Y amino acid exchange (Figure 15, SEQ ID NO: 31).
Design and cloning of vector constructs
1 . pZFNn ptl l/LbCpfl _tDT -crRNA_mGFP#1 +2+3_GFPtoYFP template
A binary vector, which does not include viral replicon sequences was used as non GVR control. The binary vector includes expression cassettes for nptll, LbCpfl and tDT-crRNA_mGFP#1 +2+3, which are directed against three target regions in mGFP5 (Figure 15, SEQ ID NO: 32). The crRNA is embedded in a Pol ll-driven RNA transcript as described by Zhong et al., 2017. In addition, the binary vector includes the repair template described in Figure 15.
2. pZFNnptll-BSCTV-Repl/LbCpf1_tDT-crRNA_mGFP#1 +2+3_GFPtoYFP template
A binary vector, which includes a nptll expression cassette and a viral replicon sequence derived from Beet severe curly top virus (BSCTV, SEQ ID NO: 33). LbCpfl and tDT- crRNA_mGFP#1+2+3 are located within the viral replicon sequence expression cassettes. The crRNA tDT-crRNA_mGFP#1 +2+3 are directed against three target regions in mGFP5-ER (Figure 16) and embedded in a Pol ll-driven RNA transcript as described by Zhong et al., 2017. In addition, the viral replicon includes the repair template described in Figure 15.
3. pZFNnptll-BSCTV-Repl/tDT-crRNA-mGFP# +2+3 -RTGFPtoYFP200-Out LbCpfl
A binary vector, which includes a nptll expression cassette and a viral replicon sequence derived from Beet severe curly top virus (BSCTV, SEQ ID NO: 34). The expression cassette for a tDT- crRNA_mGFP#1 +2+3 (Figure 16) and the repair template (Figure 15) are located within the viral replicon. The GVR construct is characterized by an LbCpfl expression cassette that is located outside the viral replicon, but still within the T-DNA.
Results and data analysis
1. Proof of functionality of the selected crRNAs in infiltrated tobacco leaves
Three crRNAs targeting mGFP5-ER have been selected. For general proof of functionality, each crRNA was cloned separately in the binary vector pZFNnptll-LbCpfl . Leaves of Nicotiana benthamiana 16c were syringe-infiltrated with these constructs and leaf samples were harvested 3 dpi and 7 dpi for DNA extraction. SDN-1 efficiencies were quantified by ddPCR. Functionality of all three crRNAs was confirmed (Figure 17). At 7 dpi all selected crRNAs showed considerable SDN-1 efficiency (4-14%).
For enabling the HDR-mediated aa exchange via a GVR-based approach, all three crRNAs were combined in a multiplex construct (tDT-crRNA-mGFP#1+2+3) and cloned into the viral replicon sequence of the binary vector pZFNnptll-BSCTV-Repl/LbCpfl (to generate the sequence in SEQ ID NO: 33) or pZFNnptll-BSCTV-Repl/Out LbCpfl (to generate the sequence in SEQ ID NO: 34).
2. Proof of HDR-based genome editing in infiltrated tobacco leaves
Leaves of Nicotiana benthamiana 16c transgenic line have been syringe infiltrated with either pZFNnptll/LbCpf1_tDT-crRNA_mGFP#1 +2+3_GFPtoYFP template (SEQ ID NO: 32), pZFNnptll- BSCTV-Repl/LbCpf1_tDT-crRNA_mGFP#1+2+3_GFPtoYFP_template (SEQ ID NO: 33) or pZFNnptll-BSCTV-Repl/tDT-crRNA-mGFPm+2+Z-RTGFPtoYFP200-Out LbCpfl (SEQ ID NO: 34). Delivery of all three constructs resulted in the conversion of GFP into YFP in a certain amounts of infiltrated tobacco cells (Figure 18). Shift from GFP to YFP fluorescence was visualized using confocal laser microscopy. GFP and YFP fluorescence was detected after excitation with 488-nm and 514-nm later light, respectively. All three binary vector constructs mediate HDR-based genome editing in tobacco. pZFN-BSCTVRepl-LbCpfl , which contains only the nuclease but no crRNA and donor repair template, was used as negative control (Figure 18 A) and do not show cells with YFP expression.

Claims

Claims
1. A method for the targeted modification of at least one genomic target sequence in a plant cell from a plant of the family Amaranthacea, preferably a cell of a plant from the genus Beta or Spinacia, more preferably a cell of a plant of the species Beta vulgaris or Spinacia oleracea, wherein the method comprises the following steps:
(a) introducing into the cell
(i) at least one viral replicon, or a sequence encoding the same, the replicon being constructed based on a Becurtovirus, or a Curtovirus genome, wherein the at least one viral replicon further comprises:
(ii) at least one genome editing system comprising at least one nucleic acid guided nuclease, nickase or an inactivated nuclease, or a sequence encoding the same, and at least one guide molecule, or a sequence encoding the same;
(iii) and, optionally at least one repairtemplate, or a sequence encoding the same;
(b) cultivating the cell under conditions allowing the expression and/or assembly of the at least one genome editing system and the at least one guide molecule and/or optionally the at least one repairtemplate; and
(c) obtaining at least one modified cell; and
(d) optionally obtaining at least one plant, plant tissue, organ, or seed regenerated from the at least one modified cell.
2. The method of claim 1 , wherein the at least one viral replicon, or the sequence encoding the same, is constructed based on a Beet curly top iran virus Becurtovirus genome, a Spinach Curly Top Arizona Virus Becurtovirus, a Beet severe curly top virus Curtovirus genome, a Beet Curly Top Virus Curtovirus genome, a Beet Mild Curly Top Virus Curtovirus genome, or a Spinach Severe Curly Top Virus Curtovirus, preferably a genome selected from SEQ ID NOs: 22, 23, 27 or 28, or a genome from a homologous virus to the respective sequences belonging to the genus Becurtovirus or Curtovirus.
3. The method of any of the preceding claims, wherein the at least one viral replicon is encoded by a sequence according to SEQ ID NO: 4 or 6, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
4. The method of any of the preceding claims, wherein the at least one viral replicon comprising the at least one genome editing system is introduced into the cell by transformation or transfection, preferably by Agrobacterium- mediated transformation, biolistic bombardment, micro- or nanoparticle delivery, or by chemical transfection.
5. The method of claim 4, wherein the at least one viral replicon comprising the at least one genome editing system is introduced into the cell by Agrobacterium-mebiateb transformation, wherein the at least one viral replicon is inserted into a binary vector, preferably wherein the binary vector comprising the at least one viral replicon has a sequence of SEQ ID NOs: 5 or 7 to 15, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
6. The method of any of the preceding claims, wherein the at least one nucleic acid guided nuclease, nickase or an inactivated nuclease, or a sequence encoding the same, of the genome editing system is selected from the group consisting of a CRISPR/Cas system, preferably from a CRISPR/Cfp1 system, a CRISPR/MAD7 system, a CRISPR/MAD2 system, a CRISPR/Cas9 system, a CRISPR/CasX system, a CRISPR/CasY system, or a CRISPR/Csm system, or any combination, variant, or catalytically active fragment thereof.
7. The method of any of the preceding claims, wherein the at least one genome editing system further comprises at least one reverse transcriptase, preferably a modified reverse transcriptase, wherein the reverse transcriptase is preferably used in combination with a nucleic acid guided nickase, and/or at least one cytidine or adenine deaminase, preferably wherein the at least one cytidine or adenine deaminase is independently selected from an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase, preferably a rat- derived APOBEC, an activation-induced cytidine deaminase (AID), an ACF1/ASE deaminase, an ADAT family deaminase, an ADAR2 deaminase, or a PmCDAI deaminase, a TadA derived deaminase, and/or a transposon, or a sequence encoding the aforementioned at least one enzyme, or any combination, variant, or catalytically active fragment thereof.
8. The method of any of the preceding claims, wherein the at least one genome editing system comprises at least one repair template, and wherein the at least one repair template comprises or encodes a double- and/or single-stranded nucleic acid sequence, and/or wherein the at least one repair template is part of the at least one guide molecule.
9. The method of claim 8, wherein the at least one repair template comprises symmetric or asymmetric homology arms and/or wherein the at least one repair template comprises at least one chemically modified base and/or backbone.
10. A plant, plant cell, tissue, organ, or seed obtainable by or obtained by a method according to any of the preceding claims.
11. A viral replicon for plant transformation, wherein the viral replicon is encoded by a sequence according to SEQ ID NO: 4 or 6, or a sequence having at least sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
12. An expression construct comprising or encoding:
(i) at least one viral replicon as defined in claim 11 , and
(ii) a vector, preferably a vector as defined in claim 5, and
(iii) at least one component of at least one a genome editing system, wherein the genome editing system comprises or encodes:
(a) at least one nucleic acid guided nuclease, nickase or an inactivated nuclease, or at least one site-directed nuclease, nickase or an inactivated nuclease, and optionally at least one nuclear or organellar localization sequence; and
(b) at least one guide molecule for guiding the at least one nucleic acid guided nuclease, nickase or an inactivated nuclease and optionally for activating at least one reverse transcriptase; and
(c) optionally at least one repair template; and
(d) optionally at least one reverse transcriptase; and
(iv) wherein the expression construct comprises or encodes at least one regulatory sequence, wherein the regulatory sequence is selected from the group consisting of a core promoter sequence, a proximal promoter sequence, a cis regulatory sequence, a trans regulatory sequence, a locus control sequence, an insulator sequence, a silencer sequence, an enhancer sequence, a terminator sequence, an intron sequence, and/or any combination thereof.
13. The expression construct of claim 12, wherein the expression construct additionally comprises or encodes at least one self-cleaving ribozyme, preferably at least one hammerhead ribozyme and/or at least one hepatitis-delta virus (HDV) ribozyme and/or at least one artichoke, sunflower or rice HDV-like ribozyme.
14. The expression construct of claim 12 or 13, wherein the expression construct additionally comprises or encodes a sequence for an RNA polymerase II promoter.
15. A use of a viral replicon according to claim 11 , or a use of an expression construct of any of claims 12 to 14, for stimulating homology-directed repair by reverting a host plant cell into the S-phase of the cell cycle, and/or for increasing homology-directed repair-based genome editing.
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