WO2019234129A1

WO2019234129A1 - Haploid induction with modified dna-repair

Info

Publication number: WO2019234129A1
Application number: PCT/EP2019/064723
Authority: WO
Inventors: Markus Niessen; Aaron HUMMEL
Original assignee: KWS SAAT SE & Co. KGaA
Priority date: 2018-06-05
Filing date: 2019-06-05
Publication date: 2019-12-12

Abstract

The present invention relates to an improved method for generating a haploid plant cell comprising at least one modification at one or more predetermined genetic location(s) as well as a method for generating a doubled haploid cell or a doubled haploid cellular system comprising at least one modification at one or more predetermined genetic location(s). Further provided is the use of a cellular system having biological activity of a haploid inducer for generating a haploid cellular system comprising at least one modification at one or more predetermined genetic location(s) or for generating a doubled haploid cellular system comprising at least one modification at one or more predetermined genetic location(s).

Description

Haploid induction with modified DNA-repair

Technical field

The present invention relates to improved methods for generating genome edited plant cells, from which any sequences encoding gene editing (GE) components have been eliminated after an editing process, which provides less off-target effects by reducing non-ho- mologous end joining (NHEJ) frequencies and increasing homology directed repair (HDR). In particular, the invention relates to methods for generating a haploid plant cell comprising at least one modification at one or more predetermined genomic location(s) by fusing a first gamete having haploid inducer activity and carrying at least one genome editing component targeted to predetermined genomic location(s) of a second gamete, with the second gamete, wherein the first gamete has an inactivated polymerase theta enzyme and preferably further inactivated DNA repair enzymes of a NHEJ pathway. Further provided are haploid and doubled haploid cellular systems comprising the at least one modification at one or more predetermined genomic location(s) as well as methods for generating doubled haploid cells or cellular systems. The invention also relates to the use of a cellular system having haploid inducer activity and carrying at least one GE component while comprising an inactivated polymerase theta enzyme and preferably further inactivated DNA repair enzymes of a NHEJ pathway for generating haploid cellular systems or doubled haploid cellular systems comprising at least one modification at one or more predetermined genomic location(s).

Background of the Invention

The generation and use of haploids is one of the most powerful biotechnological means to improve cultivated plants. The advantage of haploids for breeders is that homozygosity can be achieved already in the first generation after dihaploidization, creating doubled haploid plants, without the need of several backcrossing generations required to obtain a high degree of homozygosity. Further, the value of haploids in plant research and breeding lies in the fact that the founder cells of doubled haploids are products of meiosis, so that resultant populations constitute pools of diverse recombinant and at the same time genetically fixed individuals. The generation of doubled haploids thus provides not only perfectly useful genetic variability to select from with regard to crop improvement, but is also a valuable means to produce mapping populations, recombinant inbreds as well as instantly homozygous mutants and transgenic lines. Haploid plants can be obtained by interspecific crosses, in which one parental genome is eliminated after fertilization. It was shown that genome elimination after fertilization could be induced by modifying a centromere protein, the centromere-specific histone CENH3 in Arabidopsis thaliana (Ravi and Chan, Haploid plants produced by centromere-mediated genome elimination, Nature, Vol. 464, 2010, 615-619). With the modified haploid inducer lines, haploid ization occurred in the progeny when a haploid inducer plant was crossed with a wild type plant. Interestingly, the haploid inducer line was stable upon selfing, suggesting that a competition between modified and wild type centromere in the developing hybrid embryo results in centromere inactivation of the inducer parent and consequently in unip- arental chromosome elimination.

It has recently been recognized that haploid induction can be combined with genome editing taking place in the early zygote after fertilization by the action of GE components encoded in the genome of the haploid inducer plant line. The GE components are targeted to act on a specific location of the genome of a target cell line that is crossed with the haploid inducer line. After elimination of the inducer line^'s genome, an edited haploid plant is obtained, which no longer contains any sequences encoding GE components. This approach opens a possibility to generate edited plants, which can be considered non-transgenic or non-genetically modified (non-GM).

WO2016030019A1 describes mutations causing an alteration of the amino acid sequence in the CATD domain of CENH3, in particular within the loopl or the a2-helix of the CATD domain, which provide the biological activity of a haploid inducer. Besides CENH3, other proteins can be altered with respect to their wild-type variant to induce haploidization upon crossing. For example, EP3159413A1 discloses that at least one mutation of the kineto- chore null2 (KNL2) protein, especially a mutation causing a substitution of an amino acid within the C-terminal region of the KNL2 protein, generates haploids upon crossing. Another example is a mutated PHOSPHOLIPASE A1 (PLA1 ) gene, also known as NOT LIKE DAD1 (NLD1 ) and MATRILINEAL (MATL) (WO 2016/075255 A1 ; Kelliher et al. (2017). MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction. Nature, 542(7639), 105.; WO 2018/158301 ). Preferably, the mutant PLA1 is combined with the enhancer gene as disclosed in EP 19 176 646.8.

In W02017004375A1 , methods are described which implement haploid induction in order to perform accelerated genome editing in plants. The method uses a plant line, which has haploid inducer capacity and encodes one or more endonucleases, which can cause a targeted mutation. This line is referred to as Haploid Inducer Line for Accelerated Genome Editing (HILAGE). Crossing a HILAGE with a targeted line causes a mutation of the targeted line and subsequent elimination of the HILAGE genome carrying the endonuclease^), thus creating a haploid non-transgenic mutated line, which can be subjected to chromosome doubling to obtain a diploid line. WO2018015956A1 and WO2018015957A1 relate to haploid inducer plant lines genetically modified with a nucleic acid molecule encoding a DNA editing agent and methods of generating a target plant comprising the crossing of a haploid inducer plant genetically modified with a nucleic acid molecule encoding a DNA editing agent with a target plant of interest, thereby generating a haploid plant having a DNA editing event of interest. WO2018052919A1 discloses methods and compositions for genome editing via haploid induction. According to WO2018052919A1 , a haploid inducer comprising at least one Genome Editing Complex (GEC) is crossed with a second plant, wherein the at least one GEC modifies a genome of the second plant, thereby generating a modified genome of the second plant. The resultant plant comprises the modified genome of the second plant but sub- stantially lacks the GEC due to elimination of the haploid inducer genome.

Genome editing strategies for targeted modifications largely rely on the introduction of single or double stranded breaks (DSBs) at specific locations in a genome using site-specific nucleases (SSNs). In eukaryotic cells, genome integrity is ensured by robust and partially redundant mechanisms for repairing DNA DSBs caused by environmental stresses and errors of cellular DNA processing machinery. In most eukaryotic cells and at most stages of the respective cell cycle, the non-homologous end-joining (NHEJ) DNA repair pathway is the dominant form of repair. A second pathway uses homologous recombination (HR) of similar DNA sequences to repair DSBs. This pathway can usually be implemented in the S and G2 stages of the cell cycle by templating from the duplicated homologous region of a paired chromosome to precisely repair the DSB. However, an artificially-provided repair template (RT) with homology to the target can also be used to repair the DSB, in a process known as homology directed repair (HDR) or gene targeting. By this strategy it is possible to introduce very precise, targeted changes in the genomes of eukaryotic cells.

Early gene targeting studies in plants revealed frequencies of homologous recombination that were so low it was effectively impossible to practice gene editing for crop improvement.

Site-specific nucleases (SSNs), which can be directed to a specific target sequence and there cause a DSB, increase gene targeting frequencies by 2-3 orders of magnitude when co-delivered together with a DNA RT (Puchta et al., Proc. Natl. Acad. Sci. USA 93:5055- 5060, 1996). However, GE in plants is still hindered by low frequency of HDR repairs compared to repairs by NHEJ which can create insertions or deletions (INDELs) in the SSN target, thereby disrupting further cutting and rendering the target in a cell unusable for gene targeting. An aspect to be critically considered for GE is thus the nature of the repair mechanism induced upon cleavage of a genomic target site of interest. As DSBs, or any DNA lesions in general are detrimental for the integrity of a genome, it is of high importance that the cellular machinery provides mechanisms of double-strand break (DSB) repair in the natural environment. Cells possess intrinsic mechanisms to attempt to repair any double- or single- stranded DNA damage. DSB repair mechanisms have been divided into two major basic types, NHEJ and HDR.

NHEJ is the dominant nuclear response in animals and plants which does not require homologous sequences, but is often error-prone and thus potentially mutagenic (Wyman C., Kanaar R. "DNA double-strand break repair: all's well that ends well", Annu. Rev. Genet., 2006, 40, 363-83). Classical- and backup-NHEJ pathways are known to rely on different mechanisms, wherein both pathways are error-prone. Repair by HDR requires homology, but those HDR pathways that use an intact chromosome to repair the broken one, i.e. double-strand break repair and synthesis-dependent strand annealing, are highly accurate. In the classical homology direcetd DSB repair pathway, the 3’ ends invade an intact homolo- gous template and then serve as a primer for DNA repair synthesis, ultimately leading to the formation of double Holliday junctions (dHJs). dHJs are four-stranded branched structures that form when elongation of the invasive strand "captures" and synthesizes DNA from the second DSB end. The individual HJs are resolved via cleavage in one of two ways. Synthesis-dependent strand annealing is conservative, and results exclusively in non- crossover events. This means that all newly synthesized sequences are present on the same molecule. Unlike the NHEJ repair pathway, following strand invasion and D loop formation in synthesis-dependent strand annealing, the newly synthesized portion of the invasive strand is displaced from the template and returned to the processed end of the noninvading strand at the other DSB end. The 3’ end of the non-invasive strand is elongated and ligated to fill the gap. There is a further pathway of HDR, called break-induced repair pathway not yet fully characterized. A central feature of this pathway is the presence of only one invasive end at a DSB that can be used for repair.

The naturally occurring NHEJ pathway, is highly efficient and straightforward as it can assist in rejoining the two ends after a DSB independent of significant homology. However, this efficiency is accompanied by the drawback that this process is error-prone and can be associated with insertions or deletions. The ubiquitously present NHEJ pathway in eukaryotic cells thus hampers targeted GE approaches.

A further challenge is the propensity for introduced RTs to integrate randomly into the ge- nome at unpredictable and uncontrollable locations. One NHEJ pathway is mediated by Polymerase Q (Polymerase theta, Pol Q, or Pol theta), encoded by the POLQ gene (e.g., for plants see: van Kregten et al., 2016, T-DNA integration in plants results from polymer- ase-O-mediated DNA repair. Nature Plants 2, Article number: 16164). Polymerase Q in mammals is an atypical A-family type polymerase with an N-terminal helicase-like domain, a large central domain harboring a Rad51 interaction motif, and a C-terminal polymerase domain capable of extending DNA strands from mismatched or even unmatched termini. DNA molecules can be randomly incorporated into eukaryotic genomes through the action of Pol Q being a low fidelity polymerase (Hogg et al., 2012. Promiscuous DNA synthesis by human DNA polymerase Q. Nucleic Acids Research, Volume 40, Issue 6, 1 March 2012, Pages 2611-2622) that is required for random integration of T-DNAs in plants. Knockout mutant plants lacking Pol Q activity are incapable of integrating T-DNA molecules during Agrobacterium tumefaciens mediated plant transformation (van Kregten et al., 2016, supra). In vitro experiments identified an evolutionarily conserved loop in the polymerase domain that is essential for synapsing DNA ends during end joining to protect the genome against gross chromosomal rearrangements (Sfeir, The FASEB Journal, vol. 30, no.1 , 2016).

In practice, frequent random integrations of RTs limit the availability of the templates for use by cells in gene targeting, and make it difficult to screen cells or plants with the desired gene targeting events from a background of more abundant random integration events. Thus, efficient gene targeting in eukaryotic cells is significantly hindered by low frequencies due to the prevalence of NHEJ-mediated DSB repair, and by the difficulty of screening for gene targeting events due to frequent random integration of the RT in many treated cells.

Considering that the crossing of haploid inducer plant lines with target plant lines usually only results in elimination of the inducer genome with a frequency of less than 50%, often less than 20 % or even less than 10% (sometimes only around 1-2% e.g. in the case of mutated CENH3 protein), it is highly important to avoid off-target effects and ensure higher frequencies of HDR during the editing process in the early zygote in order obtain sufficient numbers of the desired edited plants, which at the same time no longer contain the editing components because genome elimination of the haploid inducer line has occurred

It was therefore an object of the present invention to provide a method to render the genome editing in a zygote formed by the fusion of a haploid inducer plant line, which carries genome editing components targeted to act on the genome of a target cell line, and a target line more efficient. It was particularly, it was an object of the present invention to increase HDR frequencies while suppressing relevant NHEJ pathways and random integration of RTs during the editing in the zygote in order to provide higher prevalence of accurately edited plants. The edited plants should be haploid so that a diploid plant, which is homozy- gous for the edit, can be obtained in one generation. At the same time, the edited plants should not comprise sequences encoding any genome editing components, and preferably be considered non-genetically modified (non-GM) plants.

Summary of the invention

According to a first aspect of the present invention, the above objectives are met by a method for generating a haploid plant cell comprising at least one modification at one or more predetermined genetic location(s), wherein the method comprises the following steps:

(a) providing a first gamete cell of a cellular system having biological activity of a haploid inducer, wherein the first gamete cell comprises

(I) an inactivated or partially inactivated Polymerase theta enzyme, or a sequence encoding the same; and preferably one or more further inactivated or partially inactivated DNA repair enzyme(s) of a NHEJ pathway, or one or more sequence(s) encoding the same; and

(II) at least one introduced site-specific nuclease, or a sequence encoding the same, the site-specific nuclease induces at least one single-strand break or at least one double-strand break at one or more predetermined locations) in the genome of a second gamete cell of a target cellular system before the first cell division or at least during the first and second cell division after gamete fusion; and optionally: at least one nucleic acid sequence of interest flanked by one or more homology sequence(s) complementary to one or more nucleic acid sequence(s) adjacent to one or more predetermined location(s) in the genetic material of the second gamete cell of the target cellular system; and (b) allowing the first gamete cell to fuse with a second gamete cell of a target cellular system to generate a zygote cell, the second gamete cell comprises the one or more predetermined genetic location(s).

In one embodiment according to the various aspects of the present invention, a method is provided, further comprising the steps:

(c) culturing the zygote cell such that

(i) the at least one site-specific nuclease and optionally the at least one nucleic acid of interest is expressed in the zygote cell to induce the at least one modification at the one or more predetermined location(s) in the ge- nome of the second gamete cell before the first cell division or at least during the first and second cell division after gamete fusion, wherein the at least one modification is effected by DNA repair, preferably through homology-directed repair using the at least one nucleic acid of interest as a repair template; and (ii) the set of chromosomes of the first gamete cell is eliminated; and

(d) obtaining a haploid cell comprising the genome of the second gamete cell of the target cellular system including the at least one modification at the one or more predetermined genetic location(s).

In another embodiment according to the various aspects of the present invention, a method is provided, wherein the at least one site-specific nuclease and optionally the at least one nucleic acid of interest is operably linked to a promoter, which is active in the early zygote, preferably before the first cell division, or at least during the first and second cell division after gamete fusion.

In a further embodiment according to the various aspects of the present invention, a method is provided wherein the promoter is independently selected from the group consisting of a (p)BdUbM O promoter (SEQ ID NO:1 ), a (p)ZmUbM promoter (SEQ ID NO:2), a (p)OsActin promoter (SEQ ID NO:3), and a single or double 35S promoter (SEQ ID NO:4), optionally including an ZmUbM intron, an BdUbM O intron and/or an Adh1 intron, (SEQ ID NOs: 5 to 1 1 ), or any combination thereof, or a sequence having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity when compared over the whole length of the re- spective sequence of any one of SEQ ID NOs: 1 to 11. Bd means originating from Brachy- podium distachyon, Zm means originating from Zea mays, Adh1 means originating from alcohol dehydrogenase-1 , and Os means originating from Oryza sativa.

The person skilled in the art will be able to readily identify further promoters which are active in the early zygote preferably before the first cell division, or at least during the first and second cell division after gamete fusion (see e.g. Slane D et al. (2014), Cell type-specific transcriptome analysis in the early Arabidopsis thaliana embryo, The Company of Biologists | Development 141 , 4831-480; Belmonte MF et al. (2013), Comprehensive development profiles of gene activity in regions and sub-regions of the Arabidopsis seed, PNAS 110(5):E435-44; Boavida LC et al (2011 ), Whole genome analysis of gene expression reveals coordinated activation of signaling and metabolic pathways during pollen-pistil interactions in Arabidopsis (Plant Physiology, 155, 2066-2080; and Abiko M et al. (2013), gene expression profiles in rice gametes and zygotes: Identification of gamete-enriched genes and up- and down-regulated genes in zygotes after fertilization, Journal of Experimental Botany 64(7), 1927-1940).

In yet a further embodiment, according to the various aspects of the present invention, a method is provided wherein the first gamete cell of the cellular system having biological activity of a haploid inducer comprises a polynucleotide which comprises a nucleotide sequence encoding for a protein, wherein the polynucleotide comprises at least one mutation causing an alteration of the amino acid sequence of the protein and said alteration confers the biological activity of a haploid inducer, wherein the protein is selected from the group consisting of: CENH3, Ku70, Ku80, KNL2, Cenp-C, PLA1 and PLA1 combined with a myosin heavy chain protein as defined further below.

In a preferred embodiment of the invention, a method is provided wherein the first gamete cell of the cellular system having biological activity of a haploid inducer comprises a polynucleotide which comprises at least one nucleotide sequence encoding for Ku70 and Ku80, wherein the polynucleotide comprises at least one mutation causing an alteration of the amino acid sequences of Ku70 and Ku80 and said alteration confers the biological activity of a haploid inducer to Ku70 and Ku80. Other potential proteins are well known in the art and are described e.g. in US 2012/0115132 A1.

In another preferred embodiment of the invention, a method is provided wherein the first gamete cell of the cellular system having biological activity of a haploid inducer comprises a polynucleotide which comprises at least one nucleotide sequence encoding for PLA1 , wherein the polynucleotide comprises at least one mutation causing an alteration of the amino acid sequences of PLA1 and said alteration confers the biological activity of a haploid inducer to PLA1. Further, the biological activity of a haploid inducer to PLA1 can be increased by the presence of a myosin heavy chain protein as defined further below in the the first gamete cell of the cellular system having biological activity of a haploid inducer. In one embodiment according to the various aspects of the present invention, a method is provided, wherein the at least one nucleic acid of interest introduced into the first gamete cell of the cellular system having biological activity of a haploid inducer is selected from the group consisting of: an exogenous gene, a modified endogenous gene, a synthetic sequence, an intronic sequence, a coding sequence or a regulatory sequence. In a further embodiment according to the various aspects of the present invention, a method is provided, wherein the at least one modification at one or more predetermined genetic location(s) in the genome of the second gamete cell results in the introduction of a trait of interest into the genetic material of the haploid cell.

In one embodiment according to the various aspects of the present invention, a method is provided, wherein the at least one modification at one or more predetermined genetic locations) in the genome of the second gamete cell further results in the introduction of a selection marker into the genetic material of the haploid cell.

In one embodiment according to the various aspects of the present invention, a method is provided, wherein the at least one modification is selected from a replacement of at least one nucleotide; a deletion of at least one nucleotide; an insertion of at least one nucleotide or any combination thereof.

In a further embodiment according to the various aspects of the present invention, a method is provided, wherein the method further comprises the step of culturing the haploid cell under conditions to obtain a haploid cellular system comprising the genome of the second gamete cell of the target cellular system including the at least one modification at the one or more predetermined genetic location(s), preferably wherein the haploid cellular system is a plant.

In one embodiment according to the various aspects of the present invention, a method is provided, wherein the cellular system having biological activity of a haploid inducer is a plant and/or the target cellular system is a plant. According to a further aspect, the present invention provides a method for generating a doubled haploid cell or a doubled haploid cellular system comprising at least one modification at one or more predetermined genetic location(s), wherein the method comprises

(a) generating a haploid cell comprising at least one modification at one or more prede- termined genetic location(s) by the method according to any of the aspects and embodiments above;

(b) doubling the set of chromosomes in the haploid cell or a cell derived therefrom; and

(c) obtaining a doubled haploid cell comprising the at least one modification at one or more predetermined genetic location(s); and (d) optionally culturing the doubled haploid cell under conditions to obtain a doubled haploid cellular system comprising the genome of the second gamete cell of the target cellular system including the at least one modification at the one or more predetermined genetic location(s), preferably wherein the doubled haploid cellular system is a plant.

In yet a further aspect, the present invention provides a haploid cellular system obtained by a method as described in any of the above aspects and embodiments.

In a further aspect, the present invention provides a doubled haploid cellular system obtained by a method as described in any of the above aspects and embodiments.

In yet another aspect, the present invention provides a cellular system having biological activity of a haploid inducer, comprising (a) an inactivated or partially inactivated Polymerase theta enzyme, or a sequence encoding the same;

(b) optionally one or more inactivated or partially inactivated DNA repair enzyme(s) of a NHEJ pathway, or one or more sequence(s) encoding the same;

(c) at least one site-specific nuclease, or a sequence encoding the same, the site-spe- cific nuclease induces a double-strand break at one or more predetermined location(s) in the genome of a target cellular system; optionally: at least one nucleic acid of interest flanked by one or more homology sequence(s) complementary to one or more nucleic acid sequence(s) adjacent to one or more predetermined location(s) in the genetic material of a target cellular system, preferably wherein the cellular system having biological activity of a haploid inducer and the target cellular system is a plant.

In another aspect, the present invention provides the use of a cellular system having biological activity of a haploid inducer according to the aspects above for generating a haploid cellular system comprising at least one modification at one or more predetermined genetic location(s) or for generating a doubled haploid cellular system comprising at least one modification at one or more predetermined genetic location(s).

Brief description of the drawings

Figure 1 shows a schematic overview for the generation of a homogenous GE edited (SDN 1 ) doubled haploid plant by using altered CENH3.

Figure 2 shows a schematic overview for the generation of a homogenous GE edited (SDN1 ) doubled haploid plant by using mutated KU70 or KU80.

Figure 3 shows a schematic overview for the generation of a homogenous GE edited doubled haploid plant by HDR with the use of altered CENH3 and a Pol theta knock-out muta- tion (or additional a knock-out of KU70, KU80, LiglV or any possible combination).

Figure 4 shows a schematic overview for the generation of a GE edited doubled haploid plant by HDR with the use of a Pol theta knock-out mutation and a knock-out of KU70 or KU80.

Figure 5 shows a schematic overview for the generation of a GE edited (SDN1 or SDN2) doubled haploid plant by using altered CENH3 and a co-edited endogenous gene as selection marker or identifiable phenotype.

Figure 6 shows a schematic overview for the generation of a GE edited (SDN1 or SDN2) doubled haploid plant by using KU70 or KU80 knock-out mutants and a co-edited endogenous gene as selection marker or identifiable phenotype. Figure 7 shows a schematic overview for the generation of a GE edited doubled haploid plant by HDR with use of an altered CENH3 and a Pol theta knock-out (or additional a knock-out of KU70, or KU80, or LiglV, or any possible combination) and a co-edited endogenous gene as selection marker or identifiable phenotype. Figure 8 shows a schematic overview for the generation of a GE edited doubled haploid plant by HDR with use of a Pol theta and KU70 or Pol theta and KU80 knock-out and a coedited endogenous gene as selection marker or identifiable phenotype.

Figure 9 shows overview of PolQ, Ku70, Ku80 and LiglV gene expression in the mutant lines N698253 (teb-2), N667884 (teb-5), N656431 (liglV), N656936 (ku70) and N677892 (ku80). Gene expression was determined by qRT-PCR using primers directed to a region not overlapping with the T-DNA insertion site. Col-0 wild type plants were used as reference. qRT-PCR data indicate that expression of PolQ, LiglV and Ku80 genes is significantly reduced in the respective mutant lines. Although Ku70 transcripts are detectable in N656936, the mutant line can be a null mutant.

Definitions

A“haploid plant cell” herein refers to a plant cell having only one set of chromosomes each one not being part of a pair. The number of chromosomes in a single set is called the haploid number, given the symbol n.“Gametes” are haploid cells, of which two combine in fertilization to form a“zygote” with n pairs of chromosomes, i.e. 2 n chromosomes in total. Each chromosome pair comprises one chromosome from each gamete, called homologous chromosomes. Cells and organisms with pairs of homologous chromosomes are“diploid”. A haploid cell or organism has a single set of unpaired chromosomes. A“doubled haploid” cell or organism is obtained when a haploid cell undergoes chromosome doubling. Therefore, doubled haploid cells are homozygous.

A“modification” at one or more predetermined genetic location(s) in the context of the present invention 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. In particular, a modification can be achieved by insertion or addition of one or more nucleotide^), or substitution or deletion of one or more nucleotide(s) of the original sequence or any combination of these.

A“cellular system” as used herein refers to cells, an organism or a part or a tissue of an organism, preferably a plant or a plant line, a plant part or a plant organ, differentiated and undifferentiated plant tissues, plant cells, seeds, and derivatives and progeny thereof. A“cellular system having biological activity of a haploid inducer” or a“haploid inducer” or a “haploid inducer line” is a cellular system, in particular a plant line having the capability to produce haploid offspring in at least 0.1 %, at least 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, preferably at least 1 %, preferably at least 2 %, preferably at least 3 %, pref- erably at least 4 %, preferably at least 5 %, preferably at least 6 %, preferably at least 7 %, preferably at least 8 %, preferably at least 9 %, most preferred at least 10 %, most preferred at least 15 %, most preferred at least 20 % of cases when crossed to a wild type plant. Since the chromosomes of the haploid inducer are eliminated, the resulting haploid progeny only comprises the chromosomes of the wild type parent and is not the result of sex- ually crossing of whole genomes.

An’’introduced” site-specific nuclease, site specific editor or site specific enzyme effecting DNA methylation, histone acetylation, histone methylation, histone ubiquitylation, histone phosphorylation, histone sumoylation, histone ribosylation or histone citrullination, refers to the respective site-specific agent, which has been functionally introduced into the cell or cellular system in a way that allows transcription and/or translation and/or the catalytic activity and/or binding activity of the agent in the cell. The introduction can be stable or transient. A stable introduction refers to the integration into the genome of the cell or cellular system, while a transient introduction means that the introduced site-specific agent can only act in the cell or cellular system for a limited time and will not be inherited to progeny of the cell or cellular system. Preferred in the context of the present invention is a stable introduction. An introduction may be achieved by transformation techniques such as biolis- tic approaches (e.g. particle bombardment), microinjection, permeabilising the cell membrane with various treatments such as electroporation or PEG treatment or Agrobacterium tumefaciens mediated transformation. An“inactivated” or“partially inactivated” enzyme has lost all of its enzymatic activity or, respectively, has a reduced enzymatic activity with respect to the wild-type enzyme. Inactivation or partial inactivation of an enzyme may be achieved by targeted mutations, which either lead to a knock-out or to an altered (reduced) activity.

A“site-specific nuclease” refers to a nuclease or an active fragment thereof, which is ca- pable to specifically recognize and cleave DNA at a certain location. This location is herein also referred to as a“predetermined location”. Such nucleases typically produce a double strand break (DSB), which is then repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). The nucleases include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR/Cas systems, including CRISPR/Cas9 systems, CRISPR/Cpf1 systems, CRISPR/C2C2 systems, CRISPR/CasX systems, CRISPR/CasY systems, CRISPR/Cmr systems, engineered homing endonucleases, recombinases, trans- posases and meganucleases, and/or any combination, variant, or catalytically active fragment thereof. A“repair template” 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.

A "base editor" as used herein refers to a protein or a fragment thereof having the same catalytic activity as the protein it is derived from, which protein or fragment thereof, alone or when provided as molecular complex, referred to as base editing complex herein, has the capacity to mediate a targeted base modification, i.e., the conversion of a base of interest resulting in a point mutation of interest. Preferably, the at least one base editor in the context of the present invention is temporarily or permanently linked to at least one site- specific effector, or optionally to a component of at least one site-specific effector complex. The linkage can be covalent and/or non-covalent.

An“enzyme effecting DNA methylation, histone acetylation, histone methylation, histone ubiquitylation, histone phosphorylation, histone sumoylation, histone ribosylation or histone citrullination” is an enzyme that catalyzes the respective covalent DNA or histone modifi- cation.

A "promoter" refers to a DNA sequence capable of controlling and/or regulating expression of a coding sequence, i.e., a gene or part thereof, to which it is“operably linked”. Promoters can have a broad spectrum of activity, but they can also have tissue or developmental stage specific activity. For example, they can be active in cells of roots, seeds and meriste- matic cells, etc. Certain promoters are specifically active in the early zygote after gamete fusion before the first cell division or during the first and second cell division.

A nucleic acid molecule that is“endogenous” to a cell or organism refers to a nucleic acid molecule that naturally occurs in the genome of this cell or organism. On the other hand, a nucleic acid molecule that is“exogenous” to a cell or organism refers to a nucleic acid molecule that does not naturally occur in this cell or organism but has been introduced by a transgenic event. Nucleic acid sequences or nucleic acid molecules disclosed herein can be "codon-optimized". "Codon optimization" implies that a DNA or RNA synthetically produced or isolated from a donor organism is adapted to the codon usage of different recipient organism to improve transcription rates, mRNA processing and/or stability, and/or translation rates, and/or subsequent protein folding of said recombinant nucleic acid in the cell or organism of interest. The skilled person is well aware of the fact that a target nucleic acid can be modified at one position due to the codon degeneracy, whereas this modification will still lead to the same amino acid sequence at that position after translation, which is achieved by codon optimization to take into consideration the species-specific codon usage of a tar- get cell or organism. In turn, nucleic acid sequences as defined herein may have a certain degree of identity to a different sequence, encoding the same protein, but having been codon optimized.

Whenever the present disclosure relates to the percentage of identity of nucleic acid or amino acid sequences to each other these values define those values as obtained by using the EMBOSS Water Pairwise Sequence Alignments (nucleotide) programme (www.ebi.ac.uk/Tools/psa/ emboss_water/nucleotide.html) nucleic acids or the EMBOSS Water Pairwise Sequence Alignments (protein) programme (www.ebi.ac.uk/Tools/psa/emboss_water/) for amino acid sequences. Alignments or sequence comparisons as used herein refer to an alignment over the whole length of two sequences compared to each other. 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. The skilled person is well aware of the fact that, for example, a sequence encoding a protein can be "codon-optimized" if the respective sequence is to be used in another organism in comparison to the original organism a molecule originates from.

Detailed Description

The present invention relates to several aspects to establish a new technology to increase the efficiency of generation of accurately genome edited plants, which do not comprise any sequences encoding genome editing components, via haploid induction. Genome editing in the zygote after fusion of a haploid inducer with a target cell line requires reduced off- target effects for the process to obtain adequate numbers of accurately edited haploid plant cells. In particular, HDR mechanisms to repair DSBs can provide the required accuracy but they compete with the more frequent NHEJ pathways. NHEJ pathways are mediated by a number of highly conserved enzymes. Knock-outs or knock-downs of any of these essential enzymes impair the ability of cells to use the NHEJ pathway. Impaired function of NHEJ tends to favor HDR as a partially compensatory mechanism to preserve a cell^'s aim to achieve chromosomal integrity in the presence of DSBs. Furthermore, if a repair template is used in the editing process, random integration by polymerase theta, reduces the avail- ability of RT for use in HDR mechanisms and complicates the identification of correctly edited plants.

Inhibited expression of POL theta and optionally one or more enzymes essential for NHEJ repair (e.g. Ku70 or Ku80 and further enzymes disclosed herein) in the haploid inducer line while performing targeted genome editing (GE) in the early zygote after fusion with a target line ensures dominance of HR-mediated DSB repair and thus allows highly accurate editing of the genome of the target line. Using this technology, it is possible to produce genome edited, non-GM plants, as the genome of the transgenic (introduced) site-specific agent is removed in the early zygote and never part of the resulting haploid plant. Additionally, the generation of edited haploid plants offers the potential to generate an homozygous edited doubled haploid plant after only one crossing step.

In a first aspect, a method for generating a haploid plant cell comprising at least one modification at one or more predetermined genetic location(s) is provided, wherein the method comprises the following steps:

(I) an inactivated or partially inactivated Polymerase theta enzyme, or a sequence encoding the same; and preferably one or more further inactivated or partially inactivated DNA repair enzyme(s) of a NHEJ pathway, or one or more sequence(s) encoding the same; and (II) at least one introduced site-specific nuclease, or a sequence encoding the same, the site-specific nuclease induces at least one single-strand break or at least one double-strand break at one or more predetermined locations) in the genome of a second gamete cell of a target cellular system before the first cell division or at least during the first and second cell division after gamete fusion; and optionally: at least one nucleic acid sequence of interest flanked by one or more homology sequence(s) complementary to one or more nucleic acid sequence(s) adjacent to one or more predeter- mined location(s) in the genetic material of the second gamete cell of the target cellular system; and

(b) allowing the first gamete cell to fuse with a second gamete cell of a target cellular system to generate a zygote cell, the second gamete cell comprises the one or more predetermined genetic location(s). In the first gamete cell having the biological activity of a haploid inducer, the polymerase theta enzyme and preferably also one or more further DNA repair enzyme(s) of a NHEJ pathway has/have been inactivated or partially inactivated. Furthermore, the first gamete cell carries at least one site-specific GE component targeted to act on the genome of a target cell line. The GE component may optionally comprise a repair template to be used in a HDR mechanism to repair a DSB introduced by a site-specific nuclease as part of the GE component. After fusion of the first gamete with a second gamete comprising the target site for the GE component, editing takes place under conditions when NHEJ pathways are completely or at least partially inactive and in case a repair template is provided to be used for HDR, random integrations are prevented or at least have a reduced frequency due to the (partial) inactivation of the polymerase theta enzyme. As a result, the editing process is highly accurate because HDR frequency is increased with respect to NHEJ compared to a scenario, in which the DNA repair enzyme(s) of a NHEJ and the polymerase theta enzyme are fully functional and active. Within the fraction of cases, where the haploid inducer activity of the first gamete results in the elimination of the genome of the first gamete, the frequency of accurately edited haploid cells is therefore much higher.

Besides a site-specific nuclease and optionally a repair template, the GE component may also comprise other editing tools such as base editors or enzymes that modify DNA or histones resulting in altered, i.e. reduced or increased expression or a shutdown of target genes. Thus, multiple editing processes in the genome of the target cell can be carried out in the early zygote.

In one embodiment according to the various aspects of the present invention, the first gamete may comprise instead of the site-specific nuclease or, more preferably in addition to the site-specific nuclease or sequence encoding the same and optionally the repair template, III) at least one introduced site-specific base editor, or a sequence encoding the same, the site-specific base editor substitutes at least one nucleotide at one or more predetermined location(s) in the genome of the second gamete cell of a target cellular system before the first cell division or at least during the first and second cell division after gamete fusion; and/or

IV) at least one introduced site-specific enzyme effecting DNA methylation, histone acetylation, histone methylation, histone ubiquitylation, histone phosphorylation, histone sumoylation, histone ribosylation or histone citrullination at one or more predetermined locations) in the genome of the target cellular system before the first cell division or at least during the first and second cell division after gamete fusion.

Inactivation of the DNA repair enzyme(s) of a NHEJ pathway and the polymerase theta enzyme may be achieved e.g. by generating cells or cellular systems, which harbor a mutation in the respective enzymes, which renders the enzyme inactive or partially inactive. Other strategies such as transient (partial) inhibition of the enzymes, e.g. by silencing mechanisms such as RNAi, may also be used in the context of the present invention to inactivate the DNA repair enzyme(s) of a NHEJ pathway and the polymerase theta enzyme.

The polymerase theta enzyme that is inactivated or partially inactivated according the various aspects and embodiments of the present invention may comprise an amino acid sequence according to SEQ ID NO: 30-32, or an amino acid 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 to the sequence set forth in SEQ ID NO: 30-32, respectively; or it may be encoded by a nucleic acid sequence according to SEQ ID NO 27-29, or a nucleic acid 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 to the sequence set forth in

SEQ ID No: 27-29, respectively.

In the various aspects and embodiments of the present invention, the site-specific nuclease may be a zinc-finger nuclease, transcription activator-like effector nuclease, CRISPR/Cas systems, including CRISPR/Cas9 systems, CRISPR/Cpf1 systems, CRISPR/C2C2 sys- terns, CRISPR/CasX systems, CRISPR/CasY systems, CRISPR/Cmr systems, CRISPR/Csm1 systems, CRISPR/MAD7 systems, engineered homing endonuclease, re- combinase, transposase and meganuclease, and/or any combination, variant, or catalyti- cally active fragment thereof. The site-specific nuclease and, optionally the repair template may be introduced stably or transiently into the first gamete. Preferably, they are stably integrated into the genome of the first gamete. Various methods of transformation are available to introduce the site-specific nuclease and, optionally the repair template into the first gamete. Examples are, but are not limited to, biolistic approaches such as particle bombardment, microinjection, per- meabilising the cell membrane with various treatments such as electroporation or PEG treatment or Agrobacterium tumefaciens mediated transformation.

A "homology sequence", if present, may be part of the at least one nucleic acid sequence of interest according to the various embodiments of the present invention, to be introduced to modify the genetic material of a target cellular system according to the present disclosure. Therefore, the at least one homology sequence is physically associated with the at least one nucleic acid sequence of interest within one molecule. As such, the homology sequence may be part of the at least one nucleic acid sequence of interest to be introduced and it may be positioned within the 5^' and/or 3^' position of the at least one nucleic acid sequence of interest, optionally including at least one spacer nucleotide, or within the at least one nucleic acid sequence of interest to be introduced. As such, the homology sequence^) mediate homology-directed repair by having complementarity to at least one region, the upstream and/or the downstream region, adjacent to the predetermined location within the genetic material of the cellular system to be modified. The at least one nucleic acid sequence of interest and the flanking one or more homology region(s) thus can have the function of a repair template (RT) nucleic acid sequence. In certain embodiments, the RT may be further associated with another DNA and/or RNA sequence as mediated by complementary base pairing. In an alternative embodiment the RT may be associated with other sequences, for example, sequences of a vector, e.g., a plasmid vector, which vector can be used to amplify the RT prior to transformation. Furthermore, the RT may also be physically associated with at least part of an amino acid component, preferably the site- specific nuclease. This configuration and association allows the availability of the RT in close physical proximity to the site of a DSB, i.e., exactly at the position a targeted GE event is to be effected to allow even higher efficiency rates. For example, the at least one RT may also be associated with at least one gRNA interacting with the at least one RT and further interacting with at least one portion of a CRISPR nuclease as site-specific nuclease.

The one or more homology region(s) will each have a certain degree of complementarity to the respective region flanking the at least one predetermined location upstream and/or downstream of the double-strand break induced by the at least one site-specific nuclease, i.e., the upstream and downstream adjacent region, respectively. Preferably, the one or more homology region(s) will hybridize to the upstream and/or downstream adjacent region under conditions of high stringency. The longer the at least one homology region, the lower the degree of complementarity may be. The complementarity is usually calculated over the whole length of the respective region of homology. In case only one homology region is present, this single homology region will usually have a higher degree of complementarity to allow hybridization. Complementarity under stringent hybridization conditions will be at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, and preferably at least 97%, at least 98%, at least 99%, or even 100%. At least in the region directly flanking a DSB induced (about 5 to 10 bp upstream and down- stream of a DSB), complementarities of at least 98%, at least 99%, and preferably 100% should be present. Notably, the degree of complementarity can also be lower than 85%. This will largely depend on the target genetic material and the complexity of the genome it is derived from, the length of the nucleic acid sequence of interest to be introduced, the length and nature of the further homology arm or flanking region, the relative position and orientation of the flanking region in relation to the site at least one DSB is induced, and the like.

The term "adjacent" or "adjacent to" as used herein in the context of the predetermined location and the one or more homology region(s) may comprise an upstream and a downstream adjacent region, or both. Therefore, the adjacent region is determined based on the genetic material of a cellular system to be modified, said material comprising the predetermined location.

A“functional fragment” of a nucleotide sequence as used herein means a segment of a nucleotide sequence which has the functionality identical or comparable to the complete nucleotide sequence from which the functional fragment originates. As such, the functional fragment may possess a nucleotide sequence which is identical or homologous to the complete nucleotide sequence over a length of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94% 96%, 97%, 98% or 99%. Furthermore, a“functional fragment” of a nucleotide sequence may also mean a segment of a nucleotide sequence which alters the functionality of the total nucleotide sequence, e.g., in the course of post-transcriptional gene silencing. As such, the functional fragment of a nucleotide sequence may include at least 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25, preferably at least 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120 or 140, more preferably at least 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000 successive nucleotides of the complete nucleotide sequence. A“functional part” of a protein means a segment of a protein, or a section of the amino acid sequence, that encodes for the protein, wherein the segment may exert functionality identical or comparable to the entire protein in a plant cell. A functional part of a protein has, over a length of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, or 99%, an identical or— under conservative and semi-conservative amino acid exchanges— similar amino acid sequence to that of the protein from which the functional part originates.

There may be an upstream and/or downstream adjacent region near the predetermined location. For site-specific nucleases (SSNs) inducing blunt double-strand breaks (DSBs), the "predetermined location" will represent the site the DSB is induced within the genetic material in the target cellular system. For SSNs leaving overhangs after DSB induction, the predetermined location means the region between the cut in the 5^' end on one strand and the 3^' end on the other strand. The adjacent regions in the case of sticky end SSNs thus may be calculated using the two different DNA strands as reference. The term "adjacent to a predetermined location" thus may imply the upstream and/or downstream nucleotide positions in a genetic material to be modified, wherein the adjacent region is defined based on the genetic material of a cellular system before inducing a DSB or modification. Based on the different mechanisms of SSNs inducing DSBs, the "predetermined location" meaning the location a modification is made in a genetic material may thus imply one specific position on the same strand for blunt DSBs, or the region on different strands between two cut sites for sticky cutting DSBs, or for nickases used as SSNs between the cut at the 5^' position in one strand and at the 3^' position in the other strand.

If present, the upstream adjacent region defines the region directly upstream of the 5^' end of the cutting site of a site-specific nuclease of interest with reference to a predetermined location before initiating a double-strand break, e.g., during targeted genome engineering. Correspondingly, a downstream adjacent region defines the region directly downstream of the 3^' end of the cutting site of a SSN of interest with reference to a predetermined location before initiating a double-strand break, e.g., during targeted genome engineering. The 5^' end and the 3^' end can be the same, depending on the site-specific nuclease of interest. In certain embodiments, it may also be favorable to design at least one homology region in a distance away from the DSB to be induced, i.e., not directly flanking the predetermined location/the DSB site. In this scenario, the genomic sequence between the predetermined location and the homology sequence (the homology arm) would be "deleted" after homologous recombination had occurred, which may be preferred for certain strategies as this allows the targeted deletion of sequences near the DSB. Different kinds of RT configuration and design are thus contemplated according to the present invention for those embodiments relying on a RT. RTs may be used to introduce site-specific mutations, or RTs may be used for the site-specific integration of nucleic acid sequences of interest, or RTs may be used to assist a targeted deletion.

A "homology sequence(s)" introduced and the corresponding "adjacent region(s)" can each have varying and different length from about 15 bp to about 15.000 bp, i.e., an upstream homology region can have a different length in comparison to a downstream homology region. Only one homology region may be present. There is no real upper limit for the length of the homology region(s), which length is rather dictated by practical and technical issues. According to certain embodiments, depending on the nature of the RT and the targeted modification to be introduced, asymmetric homology regions may be preferred, i.e., homology regions, wherein the upstream and downstream flanking regions have varying length. In certain embodiments, only one upstream and downstream flanking region may be pre- sent.

Based on the above, a "predetermined location" according to the present invention means the location or site in a genetic material in a cellular system, or within a genome of a cell of interest to be modified, where a targeted edit or modification is to be introduced. In certain embodiments, the predetermined location may thus coincide with the DSB induced by the at least one site-specific nuclease, wherein in other embodiment, the predetermined location may comprise the site of the DSB induced without directly aligning with the cut sites of the at least one site-specific nuclease. In yet a further embodiment, the predetermined location may be away from, i.e., at a certain distance to the DSB site. Depending on the nature of the modification to be introduced this may be the case for an embodiment, wherein a RT is used comprising at least one homology region aligning at a certain distance from the site of a DSB induced, or spanning the DSB site, and not directly aligning with the upstream and the downstream region of an induced DSB.

In one embodiment, the method described above may further comprise the steps:

(c) culturing the zygote cell such that (i) the at least one site-specific nuclease and optionally the at least one nucleic acid of interest is expressed in the zygote cell to induce the at least one modification at the one or more predetermined location(s) in the genome of the second gamete cell before the first cell division or at least during the first and second cell division after gamete fusion, wherein the at least one modification is effected by DNA repair, preferably through homology-directed repair using the at least one nucleic acid of interest as a repair template; and (ii) the set of chromosomes of the first gamete cell is eliminated; and

In case the GE component comprises one or more base editors and or DNA/histone mod- ifying enzymes, alternatively or in addition to the site-specific nuclease and, optionally the repair template,

(ia) the at least one site-specific base editor is expressed in the zygote cell to induce the at least one modification at the one or more predetermined location(s) in the genome of the second gamete cell before the first cell division or at least during the first and second cell division after gamete fusion; and/or

(ib) the at least one site-specific enzyme effecting DNA methylation, histone acetylation, histone methylation, histone ubiquitylation, histone phosphorylation, histone sumoylation, histone ribosylation or histone citrullination is expressed in the zygote cell to induce the at least one modification at one or more predetermined location(s) in the genome of the sec- ond gamete cell before the first cell division or at least during the first and second cell division after gamete fusion.

To ensure that the respective site-specific GE components (including the optional repair template(s)) are expressed in the early zygote after fusion of the first and second gamete and perform the editing before the first cell division or at least during the first and second cell division, the GE components may be under the control of certain promoters, which are active this stage. Even after the genome of the fist gamete having haploid inducer activity is eliminated from the zygote, the expressed GE components can still perform editing of the genome of the second gamete.

In one embodiment, in the method according to any of the embodiments described above, the at least one site-specific nuclease and optionally the at least one nucleic acid of interest may be operably linked to a promoter, which is active in the early zygote, preferably before the first cell division, or at least during the first and second cell division after gamete fusion. The same applies accordingly for the at least one site-specific base editor and/or the at least one site-specific enzyme effecting DNA methylation, histone acetylation, histone methylation, histone ubiquitylation, histone phosphorylation, histone sumoylation, histone ribosylation or histone citrullination, if present. In case that more than one GE components (including optional repair template(s)) have been introduced in the first gamete, the promoter for each GE component can be selected independently.

In a further embodiment according to the various aspects of the present invention, the promoters) may be selected from the group consisting of a (p)BdUbM O promoter (SEQ ID NO: 1 ), a (p)ZmUbM promoter (SEQ ID NO:2), a (p)OsActin promoter (SEQ ID NO:3), and a single or double 35S promoter (SEQ ID NO:4), optionally including an ZmUbM intron, an BdUbM O intron and/or an Adh1 intron, (SEQ ID NOs: 5 to 1 1 ), or any combination thereof, or a sequence having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity when compared over the whole length of the respective sequence of any one of SEQ ID NOs: 1 to 1 1. Bd means originating from Brachypodium distachyon, Zm means originating from Zea mays, Adh1 means originating from alcohol dehydrogenase-1 , and Os means originating from Oryza sativa. The promoter(s) may comprise a nucleic acid sequence according to SEQ ID NO: 1-1 1 , or a nucleic acid 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 to the sequence set forth in SEQ ID NO: 1-1 1 respectively.

The person skilled in the art will be able to readily identify further promoters which are active in the early zygote, preferably before the first cell division or at least during the first and second cell division after gamete fusion (see e.g. Slane D et al. (2014), Cell type-specific transcriptome analysis in the early Arabidopsis thaliana embryo, The Company of Biologists | Development 141 , 4831-480; Belmonte MF et al. (2013), Comprehensive development profiles of gene activity in regions and sub-regions of the Arabidopsis seed, PNAS 1 10(5):E435-44; Boavida LC et al (201 1 ), Whole genome analysis of gene expression reveals coordinated activation of signaling and metabolic pathways during pollen-pistil inter- actions in Arabidopsis (Plant Physiology, 155, 2066-2080; and Abiko M et al. (2013), gene expression profiles in rice gametes and zygotes: Identification of gamete-enriched genes and up- and down-regulated genes in zygotes after fertilization, Journal of Experimental Botany 64(7), 1927-1940). In one embodiment according to the various aspects of the present invention, the first gamete cell of the cellular system having biological activity of a haploid inducer may comprise a polynucleotide which comprises a nucleotide sequence encoding for a protein, wherein the polynucleotide comprises at least one mutation causing an alteration of the amino acid sequence of the protein and said alteration confers the biological activity of a haploid inducer, wherein the protein is selected from the group consisting of: CENH3, Ku70, Ku80, KNL2, Cenp-C, PLA1 and PLA1 combined with a myosin heavy chain protein as defined further below.

In a preferred embodiment of the invention, a method is provided wherein the first gamete cell of the cellular system having biological activity of a haploid inducer comprises a polynucleotide which comprises at least one nucleotide sequence encoding for Ku70 and Ku80, wherein the polynucleotide comprises at least one mutation causing an alteration of the amino acid sequences of Ku70 and Ku80 and said alteration confers the biological activity of a haploid inducer to Ku70 and Ku80. Other potential proteins are well known in the art and are described e.g. in US 2012/01 15132 Al .The nucleotide sequence encoding for a protein and comprising at least one mutation causing an alteration of the amino acid sequence of the protein and said alteration confers the biological activity of a haploid inducer, wherein the protein is selected from the group consisting of: CENH3, Ku70, Ku80, KNL2, Cenp-C may comprise a nucleic acid sequence according to SEQ ID NO: 12-21 , 24-26, or a nucleic acid 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 to the sequence set forth in SEQ ID NO: 12-21 , 24-26 respectively and the amino acid sequence of the protein, which confers the biological activity of a haploid inducer, may comprise an amino acid sequence according to SEQ ID NO: 33-42, 45- 47, or an amino acid 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 to the sequence set forth in SEQ ID NO: 33-42, 45- 47, respectively.

In one embodiment, the first gamete cell of the cellular system having biological activity of a haploid inducer may comprise a polynucleotide which comprises a nucleotide sequence encoding for a protein, wherein the polynucleotide comprises at least one mutation causing an alteration of the amino acid sequence of the protein and said alteration confers the biological activity of a haploid inducer, wherein the protein is Ku70 and/or Ku80. In another preferred embodiment of the invention, a method is provided wherein the first gamete cell of the cellular system having biological activity of a haploid inducer comprises a polynucleotide which comprises at least one nucleotide sequence encoding for PLA1 , wherein the polynucleotide comprises at least one mutation causing an alteration of the amino acid sequences of PLA1 and said alteration confers the biological activity of a haploid inducer to PLA1 . The nucleotide sequence encoding for the PLA1 protein and comprising at least one mutation causes an alteration of the amino acid sequence of the protein and said alteration confers the biological activity of a haploid inducer, wherein said PLA1 sequence

(i) having the nucleotide sequence of SEQ ID NO: 1 17 or 120 or a sequence which is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 17 or 120,

(ii) having the coding sequence of SEQ ID NO: 1 18 or 121 or a sequence which is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 18 or 121 , or

(iii) encoding the amino acid sequence of SEQ ID NO: 1 19, 122 or 123 or a sequence which is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 19, 122 or 123, and wherein the at least one mutation is preferably wherein the mutation is

(A) a deletion or mutation in SEQ ID NO: 1 17, 1 18, 120 or 121 , preferably which leads to an early stop codon causing a shortening of the encoded amino acid sequence, wherein preferably the amino acid sequence at the C-terminal end is shortened by 5 or more amino acids, and/or

(B) a deletion or mutation in SEQ ID NO: 120 or 121 preferably resulting in an amino acid sequence having SEQ ID NO: 1 13, or a deletion in SEQ ID NO: 1 17 or 1 18 preferably resulting in an amino acid sequence having SEQ ID NO: 1 16;

(C) an alteration in the coding sequence of SEQ ID NO: 1 18 or 48 which causes an amino acid exchange between amino acid positions 57 and 289 of SEQ ID NO: 1 19, 122, or 123;

(D) causes an amino acid exchange in the amino acid sequence of SEQ ID NO: 1 19, 122 or 123 at position 58, wherein arginine is replaced by glutamine (R58Q);

(E) causes an amino acid exchange in the amino acid sequence of SEQ ID NO: 1 19, 122 or 123 at position 74, wherein aspartate is replaced by asparagine (D74N);

(F) causes an amino acid exchange in the amino acid sequence of SEQ ID NO: 1 19, 122 or 123 at position 78, wherein glycine is replaced by arginine (G78R);

(G) causes an amino acid exchange in the amino acid sequence of SEQ ID NO: 1 19, 122 or 123 at position 160, wherein valine is replaced by isoleucine (V160I); (H) causes an amino acid exchange in the amino acid sequence of SEQ ID NO: 1 19, 122 or 123 at position 288, wherein serine is replaced by leucine (S288L); and/or

(I) is a knock-out mutation. More preferably the nucleotide sequence or the encoded amino acid sequence is set forth in any of SEQ ID NO: 1 14-1 16 or SEQ ID NO: 1 1 1-1 13. Further, the biological activity of a haploid inducer to PLA1 can be increased by the presence of a myosin heavy chain protein in the first gamete cell of the cellular system having biological activity of a haploid inducer. Preferably the nucleic acid molecule encoding the myosin heavy chain protein comprises a nucleotide sequence, which:

(i) is a sequence of SEQ ID NOs: 108, or a functional fragment thereof;

(ii) has a coding sequence of SEQ ID NOs: 109; or

(iii) is complementary to the sequence from (i) or (ii); or

(iv) is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence from (i) or (ii); or

(v) encodes a protein comprising an amino acid sequence selected from the group con- sisting of SEQ ID NOs: 1 10, or a functional part of the protein; or

(vi) encodes a protein comprising an amino acid sequence which is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 10, or a functional part of the protein; or

(vii) hybridizes with a sequence from (iii) under stringent conditions.

Ku70 and Ku80 knockout mutants can confer haploid inducer activity and at the same time they represent DNA repair enzyme(s) of a NHEJ pathway. Therefore, if the first gamete comprises inactivated or partially inactivated KU70 and/or Ku80, it has haploid inducer activity and a reduced frequency of NHEJ events. A single knockout can thus provide a first gamete with two required properties. In contrast, mutants of other proteins that may confer haploid inducer activity, such as CENH3, have to be identified by introducing point mutations and screening for the desired haploid inducer activity while maintaining the function of the protein, because a loss of function would be lethal for the cell.

In one embodiment according to the various aspects of the present invention, the one or more further DNA repair enzyme(s) of a NHEJ pathway to be inactivated or partially inactivated may be independently selected from the group consisting of Ku70, Ku80, DNA- dependent protein kinase, Ataxia telangiectasia mutated (ATM), ATM - and Rad3 - related (ATR), Artemis, XRCC4, DNA ligase IV (LiglV) and XLF, or any combination thereof. In one embodiment according to the various aspects of the present invention, at least two, at least three, or at least four further DNA repair enzymes of a NHEJ pathway may be inactivated or partially inactivated.

In one embodiment according to the various aspects of the present invention, the one or more further DNA repair enzyme(s) of a NHEJ pathway to be inactivated or partially inactivated may be Ku70, or a nucleic acid sequence encoding the same, wherein the Ku70 may comprise an amino acid sequence according to SEQ ID NO: 36-38, or an amino acid 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% se- quence identity to the sequence set forth in SEQ ID NO: 36-38, respectively, or the nucleic acid sequence encoding the same may comprise a sequence according to SEQ ID NO: 15-17, or a nucleic acid 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 to the sequence set forth in SEQ ID NO: 15-17, re- spectively.

In a further embodiment, the one or more further DNA repair enzyme(s) of a NHEJ pathway to be inactivated or partially inactivated may be Ku80, or a nucleic acid sequence encoding the same, wherein the Ku80 may comprise an amino acid sequence according to SEQ ID NO: 39-41 , or an amino acid 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 to the sequence set forth in SEQ ID NO: 39-41 , respectively, or the nucleic acid sequence encoding the same may comprise a sequence according to SEQ ID NO: 18-20, or a nucleic acid 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 to the sequence set forth in SEQ ID NO: 18-20, respectively.

In a further embodiment, the one or more further DNA repair enzyme(s) of a NHEJ pathway to be inactivated or partially inactivated may be DNA-dependent protein kinase, or a nucleic acid sequence encoding the same, the DNA-dependent protein kinase may comprise an amino acid sequence according to SEQ ID NO: 48-50, or an amino acid 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 to the sequence set forth in SEQ ID NO: 48-50, respectively, or the nucleic acid sequence encoding the same may comprise a sequence according to SEQ ID NO: 51-53, or a nucleic acid 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 to the sequence set forth in SEQ ID NO: 51 -53, respectively.

In yet a further embodiment, the one or more further DNA repair enzyme(s) of a NHEJ pathway to be inactivated or partially inactivated may be ATM, or a nucleic acid sequence encoding the same, the ATM may comprise an amino acid sequence according to SEQ ID NO: 54-56, or an amino acid 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 to the sequence set forth in SEQ ID NO: 54-56, respectively, or the nucleic acid sequence encoding the same may comprise a sequence according to SEQ ID NO: 57-59, or a nucleic acid 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 to the sequence set forth in SEQ ID NO:57-59, respectively. In still a further embodiment, the one or more further DNA repair enzyme(s) of a NHEJ pathway to be inactivated or partially inactivated may be ATM - and Rad3 - related (ATR), or a nucleic acid sequence encoding the same, the ATR may comprise an amino acid sequence according to SEQ ID NO: 60-62, or an amino acid 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 to the sequence set forth in SEQ ID NO: 60-62, respectively, or the nucleic acid sequence encoding the same may comprise a sequence according to SEQ ID NO: 63-65, or a nucleic acid 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% se- quence identity to the sequence set forth in SEQ ID NO: 63-65, respectively.

In a further embodiment, the one or more further DNA repair enzyme(s) of a NHEJ pathway to be inactivated or partially inactivated may be Artemis, or a nucleic acid sequence encoding the same, the Artemis may comprise an amino acid sequence according to SEQ ID NO: 66-68, or an amino acid 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 to the sequence set forth in SEQ ID NO: 66-68, respectively, or the nucleic acid sequence encoding the same may comprise a sequence according to SEQ ID NO: 69-71 , or a nucleic acid 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 to the sequence set forth in SEQ ID NO 69-71 , respectively.

In another embodiment, the one or more further DNA repair enzyme(s) of a NHEJ pathway to be inactivated or partially inactivated may be XRCC4, or a nucleic acid sequence encod- ing the same, the XRCC4 may comprise an amino acid sequence according to SEQ ID NO: 72-74, or an amino acid 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 to the sequence set forth in SEQ ID NO: 72-74, respectively, or the nucleic acid sequence encoding the same may comprise a sequence according to SEQ ID NO: 75-77, or a nucleic acid 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 to the sequence set forth in SEQ ID NO: 75-77, respectively.

In a further embodiment, the one or more further DNA repair enzyme(s) of a NHEJ pathway to be inactivated or partially inactivated may be DNA ligase IV, or a nucleic acid sequence encoding the same, the DNA ligase IV may comprise an amino acid sequence according to SEQ ID NO: 78-80, or an amino acid 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 to the sequence set forth in SEQ ID NO: 78-80, respectively, or the nucleic acid sequence encoding the same may comprise a sequence according to SEQ ID NO: 81-83, or a nucleic acid 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 to the sequence set forth in SEQ ID NO: 81-83, respectively. In still another embodiment, the one or more further DNA repair enzyme(s) of a NHEJ pathway to be inactivated or partially inactivated may be XLF, or a nucleic acid sequence encoding the same.

In one embodiment according to the various aspects of the present invention, the at least one nucleic acid sequence of interest introduced into the first gamete cell of the cellular system having biological activity of a haploid inducer may be selected from the group consisting of: an exogenous gene, a modified endogenous gene, a synthetic sequence, an intronic sequence, a coding sequence or a regulatory sequence. The exogenous or endogenous gene or the coding sequence may be exogenous or endogenous to either or both, the cellular system from which the first gamete is derived and the target cellular system from which the second gamete is derived. The edit may thus result in the integration of a transgene or in the modification of an endogenous gene in the genome of the target cell or cellular system.

In one embodiment, a regulatory sequence according to the present invention may be a promoter sequence, wherein the editing or mutation or modulation of the promoter comprises replacing the promoter, or promoter fragment with a different promoter (also referred to as replacement promoter) or promoter fragment (also referred to as replacement pro- moter fragment), wherein the promoter replacement results in any one of the following or any one combination of the following: an increased promoter activity, an increased promoter tissue specificity, a decreased promoter activity, a decreased promoter tissue specificity, a new promoter activity, an inducible promoter activity, an extended window of gene expression, a modification of the timing or developmental progress of gene expression in the same cell layer or other cell layer, for example, extending the timing of gene expression in the tapetum of anthers, a mutation of DNA binding elements and/or a deletion or addition of DNA binding elements. The promoter (or promoter fragment) to be modified can be a promoter (or promoter fragment) that is endogenous, artificial, pre-existing, or transgenic to the target cell that is being edited. The replacement promoter or fragment thereof can be a promoter or fragment thereof that is endogenous, artificial, pre-existing, or transgenic to the target cell that is being edited. Any other regulatory sequence according to the present disclosure may be modified as detailed for a promoter or promoter fragment above.

In still another embodiment according to the various aspects of the present invention, the at least one nucleic acid sequence of interest may be a transgene, or part of a transgene, of an organism of interest, wherein the transgene or part of the transgene is selected from the group consisting of a gene encoding resistance or tolerance to abiotic stress, including drought stress, osmotic stress, heat stress, cold stress, oxidative stress, heavy metal stress, nitrogen deficiency, phosphate deficiency, salt stress or waterlogging, herbicide resistance, including resistance to glyphosate, glufosinate/phosphinotricin, hygromycin, pro- toporphyrinogen oxidase (PPO) inhibitors, ALS inhibitors, and Dicamba, a gene encoding resistance or tolerance to biotic stress, including a viral resistance gene, a fungal resistance gene, a bacterial resistance gene, an insect resistance gene, or a gene encoding a yield related trait, including lodging resistance, flowering time, shattering resistance, seed color, endosperm composition, or nutritional content. In another embodiment according to the various aspects of the present invention, the at least one nucleic acid sequence of interest may be at least part of a modified endogenous gene of an organism of interest, wherein the modified endogenous gene comprises at least one deletion, insertion and/or substitution of at least one nucleotide in comparison to the nucleic acid sequence of the unmodified endogenous gene.

In still another embodiment according to the various aspects of the present invention, the at least one nucleic acid sequence of interest may be at least part of a modified endogenous gene of an organism of interest, wherein the modified endogenous gene comprises at least one of a truncation, duplication, substitution and/or deletion of at least one nucleic acid position encoding a domain of the modified endogenous gene.

In yet another embodiment according to the various aspects of the present invention, the at least one nucleic acid sequence of interest may be at least part of a regulatory sequence, wherein the regulatory sequence comprises at least one of a core promoter sequence, a proximal promoter sequence, a cis regulatory sequence, a trans regulatory sequence, a locus control sequences, an insulator sequence, a silencer sequence, an enhancer sequence, a terminator sequence, and/or any combination thereof.

In one embodiment according to the various aspects of the present invention, the at least one modification at one or more predetermined genetic location(s) in the genome of the second gamete cell results in the introduction of a trait of interest into the genetic material of the haploid cell.

Non-limiting examples of traits that can be introduced by the method according to this embodiment are resistance or tolerance to insect pests, such as to rootworms, stem borers, cutworms, beetles, aphids, leafhoppers, weevils, mites and stinkbugs. These could be made by modification of plant genes, for example, to increase the inherent resistance of a plant to insect pests or to reduce its attractiveness to said pests. Other traits can be resistance or tolerance to nematodes, bacterial, fungal or viral pathogens or their vectors. Still other traits could be more efficient nutrient use, such as enhanced nitrogen use, improvements or introductions of efficiency in nitrogen fixation, enhanced photosynthetic efficiency, such as conversion of C3 plants to C4. Yet other traits could be enhanced toler- ance to abiotic stressors such as temperature, water supply, salinity, pH, tolerance for extremes in sunlight exposure. Additional traits can be characteristics related to taste, appearance, nutrient or vitamin profiles of edible or feedable portions of the plant, or can be related to the storage longevity or quality of these portions. Finally, traits can be related to agronomic qualities such resistance to lodging, shattering, flowering time, ripening, emergence, harvesting, plant structure, vigor, size, yield, and other characteristics.

In one embodiment according to the various aspects of the present invention, the at least one modification at one or more predetermined genetic location(s) in the genome of the second gamete cell further results in the introduction of a selection marker into the genetic material of the haploid cell.

Edited plants can be easily identified and separated from non-edited plant, when they are co-edited with selectable markers. Based on specific resistance or visual markers, screenings can be performed. Any endogenous gene which could be modified in a convenient way which confirms either a resistance, or a phenotypic marker (shape, color, fluorescence etc.) could be used. Phenotypic examples in corn might be e.g. glossy genes, golden, zebra7/lemonwhite1 , tiedyed, nitrate reductase family members (for corn and sugar beet) etc..

In one embodiment according to the various aspects of the present invention, the at least one modification is selected from a replacement of at least one nucleotide; a deletion of at least one nucleotide; an insertion of at least one nucleotide.

Using site-specific nuclease(s) as part of the GE component allows to introduce a DSB in one or more predetermined location(s) of the genome of the target cell. These can be repaired without a repair template leading to an insertion or deletion (InDel) of one or more nucleotides (SDN1 event), with a repair template leading to InDEIs of max. 20 base pairs or base exchanges of 1-2 bases (SDN2 event), or with a repair template to insert/replace large fragments or complete genes (SDN3 event).

Alternatively, or in addition, a modification may be a change of the DNA methylation; a change in histone acetylation, histone methylation, histone ubiquitylation, histone phos- phorylation, histone sumoylation, histone ribosylation or histone citrullination or any combination thereof.

In a further embodiment according to the various aspect of the present invention, the method further comprises the step of culturing the haploid cell under conditions to obtain a haploid cellular system comprising the genome of the second gamete cell of the target cellular system including the at least one modification at the one or more predetermined genetic location(s), preferably wherein the haploid cellular system is a plant. An edited haploid cell of a target cellular system, from which the genome of the haploid inducer cell has been eliminated can be cultured to obtain the target cellular system having the desired modification in its genome but lacking any GE components.

In on embodiment, the cellular system having biological activity of a haploid inducer is a plant and/or the target cellular system is a plant.

In any embodiment according to the various aspects of the present invention, the plant may be a plant species selected from the group consisting of: Hordeum vulgare, Hordeum bul- busom, Sorghum bicolor, Saccharum officinarium, Zea mays, Setaria italica, Oryza minuta, Oriza sativa, Oryza australiensis, Oryza alia, Triticum aestivum, Secale cereale, Malus do- mestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus glochid- iatus, Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus gran- dis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Solarium lycoper- sicum, Solarium tuberosum, Coffea canephora, Vitis vinifera, Erythrante guttata, Genlisea aurea, Cucumis sativus, Morns notabilis, Arabidopsis arenosa, Arabidopsis lyrata, Ara- bidopsis thaliana, Crucihimalaya himalaica, Crucihimalaya wallichii, Card amine flexuosa, Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsis pumila, Arabis hirsute, Bras- sica napus, Brassica oeleracia, Brassica rapa, Raphanus sativus, Brassica juncea, Bras- sica nigra, Eruca vesicaria subsp. sativa, Citrus sinensis, Jatropha curcas, Populus tricho- carpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicer arietinum, Cicer retic- ulatum, Cicer judaicum, Cajanus cajanifolius, Cajanus scarabaeoides, Phaseolus vulgaris, Glycine max, Astragalus sinicus, Lotus japonicas, Torenia fournieri, Allium cepa, Allium istulosum, Allium sativum, and Allium tuberosum.

In one aspect, the present invention provides a method for generating a doubled haploid cell or a doubled haploid cellular system comprising at least one modification at one or more predetermined genetic location(s), wherein the method comprises

(a) generating a haploid cell comprising at least one modification at one or more predetermined genetic location(s) by any embodiment of the method described above;

(c) obtaining a doubled haploid cell comprising the at least one modification at one or more predetermined genetic location(s); and

(d) optionally culturing the doubled haploid cell under conditions to obtain a doubled haploid cellular system comprising the genome of the second gamete cell of the target cellular system including the at least one modification at the one or more predetermined genetic location(s), preferably wherein the doubled haploid cellular system is a plant.

Untreated haploid plants are nearly completely sterile. Upon doubling the set of chromosomes in the haploid cell obtained by any embodiment of the method described above, a doubled haploid cell having the genome of the target cellular system and being homozygous for the at least one modification can be generated. Such a cell can be cultured into a cellular system. Doubling of the set of chromosomes may be achieved by colchicine treatment or spontaneous chromosome doubling.

In one aspect, the present invention relates to a haploid cellular system obtained by a method according to any of the embodiments described above.

In a further aspect, the present invention relates to a doubled haploid cellular system obtained by a method according to any of the embodiments described above.

In one aspect, the present invention provides a cellular system having biological activity of a haploid inducer, comprising (a) an inactivated or partially inactivated Polymerase theta enzyme, or a sequence encoding the same;

Alternatively, or preferably in addition, the cellular system having biological activity of a haploid inducer comprises:

(d) at least one site-specific base editor, or a sequence encoding the same, the site- specific base editor substitutes at least one nucleotide at one or more predetermined locations) in the genome of a target cellular system; and/or (e) at least one site-specific enzyme effecting DNA methylation, histone acetylation, histone methylation, histone ubiquitylation, histone phosphorylation, histone sumoylation, histone ribosylation or histone citrullination at one or more predetermined location(s) in the genome of a target cellular system. For the cellular system, the features and variations described in the context of the aspects and embodiments detailed above apply accordingly.

In yet a further aspect, the present invention relates to the use of a cellular system having biological activity of a haploid inducer as described above for generating a haploid cellular system comprising at least one modification at one or more predetermined genetic loca- tion(s) or for generating a doubled haploid cellular system comprising at least one modification at one or more predetermined genetic location(s).

A cellular system having biological activity of a haploid inducer as described above allows the generation of an accurately edited haploid target cell or cellular system, which does not comprise any sequences encoding GE components including repair templates. Doubled haploid target cells or cellular systems can further be obtained which are homozygous for the edit.

Example 1 : Design of gene editing plasmids for use with haploid-inducing lines

To prepare for gene editing experiments, T-DNA plasmids were constructed for efficient CRISPR-based editing in Arabidopsis using the floral dip method (Clough SJ and Bent AF, 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735-43.) to introduce the nuclease into the haploid inducer line (SEQ ID NO: 84, 85 and 86).

These plasmid vectors contained a previously described Cas9 (SEQ ID NO: 87) codon- optimized for the expression in Arabidopsis (Fauser, F., Schiml, S. and Puchta, H. (2014), Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J, 79: 348-359. doi: 10.1 1 1 1/tpj.12554) that was expressed from a promoter that is active in the early cell cycles of the fertilized embryo SEQ ID NO: 88). The plasmid also contained one or two U6 RNA polymerase III promoters (SEQ ID NO: 89) for expression of one or two single guide RNA (sgRNA) (SEQ ID NO: 90, 91 and 92) component of the CRISPR nuclease.

Alternatively, any other CRISPR machinery like Cpf1 (codon-optimized for Arabidopsis thaliana) may be used for the approach (SEQ ID NO: 93). In addition to the CRISPR nuclease components, the plasmid also contains a fluorescent reporter gene, tdTomato (SEQ ID NO: 94) (the tdTomato fluorescence represents any visible phenotype that can be delivered via transgenesis), driven by a strong constitutive promoter (SEQ ID NO: 1-11 ). Finally, a selectable marker cassette expressing the bar gene is also included (SEQ ID NO: 95). All these elements are contained within the T-DNA (SEQ ID NO: 84, 85 and 86) so they are transferred into the plant cells after the floral dip.

Example 2: Transformation of Arabidopsis haploid-inducing lines with gene editing vectors Plant growth:

Arabidopsis plants were grown to flowering stage at 24°C day /20°C night, with 250 pmol m-2 s-1 light intensity, i.e. at the beginning in short day, 8hrs light, shift for flower induction after 5 weeks to 16 hrs light.

Unless otherwise noted, the plants used were Arabidopsis ecotype Col-0 plants To obtain more floral buds per plant, inflorescences were clipped after most plants had formed primary bolts, relieving apical dominance and encouraging synchronized emergence of multiple secondary bolts.

Plants were infiltrated or dipped when most secondary inflorescences were about 1-10 cm tall (4-8 days after clipping).

Standard protocols for culture of Agrobacterium tumefaciens and inoculation of plants:

Agrobacterium tumefaciens strain AGL1 was used in all experiments - Bacteria were grown to stationary phase in liquid culture at 28°C, 250 r.p.m. in sterilized LB (10 g tryptone, 5 g yeast extract, 5 g NaCI per litre water)

Cells were harvested by centrifugation for 20 min at room temperature at 5500 g and then resuspended in infiltration medium to a final OD600 of approximately 0.80 prior to use.

The revised floral dip inoculation medium contained 5.0% sucrose and 0.04% Silwet L-77 silicone surfactant (www.momentive.com).

For floral dip, the inoculum was added to a beaker, plants were inverted into this suspension such that all above-ground tissues were submerged, and plants were then removed after 2-3 min and the procedure was reproduced twice.

Dipped plants were removed from the beaker, placed in a plastic tray and covered with a tall clear-plastic dome to maintain humidity.

Plants were left in a dark location overnight at 16 - 18 °C and returned to the light the next day. Plants were grown for a further 3-5 weeks until siliques were brown and dry.

Seeds were harvested.

Modified according to Clough et al, supra.

Arabidopsis seeds transformed by the above protocol are germinated on media containing basta (i.e. phosphinotricin or glufosinate) to select for seedlings harboring an integrated T- DNA. Resistant seedlings are checked for tdTomato fluorescence to verify the presence of the T-DNA per se. Example 3: Phenotypic markers

To use phenotypic genes as indicators to increase the efficiency of screening and recovering desirable plants during gene editing in haploid induction experiments, sgRNAs targeted to two phenotypic genes, Adh1 and TT4 (SEQ ID NO: 90, 91 and 92) (Fauser, F., Schiml, S. and Puchta, H. (2014), Both CRISPR/Cas-based nucleases and nickases may be used efficiently for genome engineering in Arabidopsis thaliana. Plant J, 79: 348-359. doi: 10.1 1 1 1 /tpj.12554), were cloned under control of the U6 promoters described above (SEQ ID NO: 89). Functional Adh1 converts allyl alcohol to acrylaldehyde, which is toxic to plants. However, loss of function Adh1 mutant plants are resistant to allyl alcohol treatment. TT4 loss of function have yellow seed coats that are distinguishable from WT seed coats due to the absence of anthocyanin production (Zangh et al. (2010), High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases. PNAS 2010 107 (26) 12028-12033).

The TT4 loss of function phenotype represents any mutant phenotype that can be generated by CRISPR-mediated mutagenesis, and the mutagenesis frequency at Adh1 meas- ured by resistance to allyl alcohol poisoning represents the frequency of co-editing of any gene of interest (e.g., a trait gene) (these gene targets are representative to demonstrate the concept and the potential of the invention). In corn for example, the phenotype delivered on the T-DNA could be eGFP or mNeonGreen fluorescence (for easy detection) (SEQ ID NO: 96), the loss of function phenotype could be due to mutagenesis of the Glossy 2 gene (SEQ ID NO: 97) which alters the cuticular wax structure and causes adherence of water droplets to the leaf, and the co-edited gene could be a trait gene such as a yield, drought, nitrogen use, resistance gene etc. that has no obvious phenotype. Plants exhibiting loss of green fluorescence and gain of glossy 2 phenotype (due to Glossy 2 loss of function) would be screened by PCR and sequencing for co-editing at the trait gene. Example 4: Crossing to produce transgene-free, edited haploid lines in a single generation Arabidopsis plants were grown to flowering stage at 24°C day/20°C night, with 250 pmol m-2 s-1 light intensity (see above)

Haploid inducer plants carrying the T-DNA described in Example 1 (for CENH3 and KU mutants (SEQ ID NO: 86)) were used either as mother plants and pollinated, or used to pollinate another plant

Plants were grown for a further 3-5 weeks until siliques were brown and dry and seeds were harvested.

Analysis of seeds and offspring population

- Seeds: status of the seeds, viability shape etc. (could be taken from

Karimi-Ashtiyani, R., Ishii, T., Niessen, M., Stein, N., Heckmann, S., Guru- shidze, M., ... & Weiss, O. (2015). Point mutation impairs centromeric CENH3 loading and induces haploid plants. Proceedings of the National Academy of Sciences, 112(36), 1121 1-11216.)

PCR-markers: in case a non Col-0 pollinator is used or a heterozygous pollinator or mother in the vise versa pollination it is possible to use specific SNP-marker to test

Flow cytometric analysis:

equal amounts of leaf material from 5-10 individuals were chopped to- gether with a sharp razor blade in nuclei isolation buffer supplemented with

DNase-free RNase (50 pg/mL) and propidium iodide (50 pg/mL). The nuclei suspensions were filtered through a 35-pm cellstrainer cap into 5-mL polystyrene tubes (BD Biosciences) and measured on a FACStarPLUS cell sorter (BD Biosciences) equipped with an argon ion laser INNOVA 90C (Coherent). Approximately 10,000 nuclei were measured and analyzed using the software CELL Guest ver. 3.3 (BD Biosciences). The resulting histograms were compared with a reference histogram of a diploid wild-type plant. If an additional peak indicated haploidy, single plants were measured again to identify the haploid individuals. A same procedure may be applied with seed material (Karimi-Ashtiyani, R et al. , supra).

Example 5: Selection of edited plants

Haploid seedlings that have undergone editing events are determined phenotypically as follows: first seeds with yellow coats (indicating TT4 loss of function) can be separated. Haploid seedlings that have acquired a TT4 loss of function mutation due to action of the CRISPR nuclease and that have also lost the T-DNA due to elimination of the haploid- inducer genome demonstrate the utility of visible phenotypes for identifying non-transgenic, haploid, gene-edited plants among thousands of progenies generated in these types of experiments. Finally, these seedlings are exposed to allyl alcohol during germination to assess the frequency of Adh1 loss of function mutations as an indication of how frequently co-editing occurs at unlinked target genes during haploid induction experiments. In this case the Adh1 represents the target gene, however it may also be used as selectable marker in case another gene (trait gene) is used as target.

In particular, first selection is on yellow seed coats resulting from loss of TT4 function. That gives the percentage of edits in TT4. To calculate how many co-edits have occurred, the seeds are put on Allyl-alcohol and the germinating plants are counted. This gives the per- centage of co-editing happening in TT4 and Adh1. This is the reference/baseline to be expected for other gene target combinations.

Example 6: Determination of edits in the genome

Determination of SDN1 (repaired double-strand break without repair template leading to InDels), SDN2 (targeted double-strand break repair with repair template, leading to InDels of max. 20bp or base exchanges of 1-20 bases), and SDN3 (targeted double-strand break with repair template to insert/replace large fragments of complete genes) events at the desired position can be made by different standard PCR and sequencing methods including NGS amplicon sequencing.

Absence of any transgenic sequence was also determined by standard PCR and sequenc- ing methods.

Example 7: Genome doubling to produce homozygous edited doubled haploid plants

Colchicine treatment is a standard method to double the genome. Colchicine treatment is applied (application of 0.25% colchicine directly dropped in the middle SAM region of the plants) before bolting greatly inhibits plant growth, but after recovery, all branches are fertile and produce diploid seeds. Nearly all plants treated with colchicine prior to bolting were successfully converted into diploids. The regeneration time was variable.

Colchicine treatment after bolting kills the primary inflorescence and secondary fertile inflorescences begin growing out. Treatment after bolting produces a lower frequency of successful somatic chromosome doubling. Example 8: Generation of homogenous GE edited (SDN1 ) doubled haploid plants by using altered CENH3 Figure 1 shows a schematic overview for the generation of a homogenous GE edited (SDN 1 ) doubled haploid plant by using altered CENH3.

Altered CENH3 variants are generated by GE based targeted mutation. Suitable mutations that result in CENH3 inducer lines have been previously described (see e.g. WO 2016/030019 and WO 2016/102665). In the first step, suitable gRNAs (SEQ ID NO: 98) are designed by using the CRISPR RGEN Tool (http://www.rgenome.net/cas-designer/ [Park J., Bae S., and Kim J.-S. Cas-Designer: A web-based tool for choice of CRISPR- Cas9 target sites. Bioinformatics 31 , 4014-4016 (2015). and Bae S., Park J., and Kim J.-S. Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473-1475 (2014)). These gRNAs are then cloned into the above described vectors comprising the gene editing constructs. In addition, a repair template (SEQ ID NO: 99) required for introducing the desired CENH3 mutation is provided. After transformation of the T DNA Plasmid, transformed plants are analyzed for the desired mutation via PCR amplification of the genomic target sequence. Plants containing the desired CENH3 mutation are self-crossed to obtain homozygous mutants.

Parent plant 1 has haploid inducer activity based on an altered CENH3 gene and also carries the GE plasmid (SEQ ID NO: 86) integrated via transformation as described above. After crossing with parent plant 2, the GE component edits the genome of parent plant 2 in the early zygote. The genome of parent plant 1 including all transgenic elements is completely removed. The haploid plant with the SDN1 event is analyzed and doubled to generate a homogenous edited doubled haploid plant.

Example 9: Generation of homogenous GE edited (SDN1 ) doubled haploid plants by using mutated KU70 or KU80 (knock-out mutation)

Ku70 and/or Ku80 knock out mutants are generated by means of GE based targeted mutations. Suitable gRNAs (SEQ ID NO: 102, 103, 104 and 105) are designed by using the CRISPR RGEN Tool (http://www.rgenome.net/cas-designer/ [Park J., Bae S., and Kim J.- S. Cas-Designer: A web-based tool for choice of CRISPR-Cas9 target sites. Bioinformatics 31 , 4014-4016 (2015). and Bae S., Park J., and Kim J.-S. Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473-1475 (2014).). These gRNAs are then cloned into the above described vectors comprising the gene editing components. After transformation of the T DNA plasmids, plants are analyzed by PCR amplification to validate the gene editing efficiency. Plants containing the desired Ku70 and/or Ku80 mutations are self-crossed to obtain homozygous Ku70 and/or Ku80 mutant plants.

Parent plant 1 has haploid inducer activity based on a KU70 or KU80 endogenous gene knock-out. Furthermore, it carries the GE plasmid (SEQ ID NO: 86) integrated via transformation as described above. After crossing with parent plant 2, the GE component edits the genome of parent plant 2 in the early zygote. Because KU70 and KU80 also represent DNA repair enzymes of a NHEJ pathway, the frequency of NHEJ event during editing may be lower. The genome of parent plant 1 including all transgenic elements is completely removed. The haploid plant with the SDN1 event is analyzed and doubled to generate a homogenous edited doubled haploid plant.

Example 10: Generation of homogenous GE edited doubled haploid plants by HDR with the use of altered CENH3 and a Pol theta knock-out mutation (or additional a knock-out of KU70, KU80, LiglV or any possible combination)

Altered CENH3 mutants are generated as described above. Pol theta knock out mutations are generated by T-DNA insertion mutant lines (commercially available, see table 1 ) or by means of GE based targeted mutation. Suitable gRNAs are identified as described above (SEQ ID NO: 106 and 107) and cloned into the above described constructs. After transfor- mation of the T-DNA plasmids, plants are analyzed by PCR amplification to validate the gene editing efficiency. Plants containing the desired Pol theta and CENH3 mutations are self-crossed to obtain homozygous mutant plants.

Table 1 : Overview of the tested mutant lines

Exemplary, to test whether double mutants of Pol Q (PolQ) and at least one mutant from the group of Ku70, Ku80 or LiglV are viable T-DNA mutant lines have been used. T-DNA insertion and expression of disrupted genes were determined by PCR / qRT-PCR (Figure 9). Next, all mutant lines were grown until flowering, and the two PolQ (At4g32700) mutants (teb-2 and teb-5) were each crossed with the Ku70 (At1g 16970), Ku80 (At1g48050) or LiglV (At5g57160) mutants to obtain the respective double mutants. Importantly, all crossings resulted in viable seeds which were harvested and propagated to F2. F2 plants were characterized by PCR for T-DNA insertion into both alleles of PolQ, Ku70, Ku80 and LiglV, respectively. For 5 of the 6 crossings, plants with T-DNA insertions into both alleles of both genes were identified. For the teb-2 x ku70 crossing, no homozygous double mutants were identified (Table 2).

Table 2: Overview of F3 generations obtained from double mutant lines.

All double mutants showed no severe growth phenotypes, even though some plants showed reduced growth. F3 seeds were harvested from these plants. None of the identified double mutants showed severe fertility defects. It was thus possible to obtain enough seeds for all double mutants.

Figure 3 shows a schematic overview for the generation of a homogenous GE edited dou- bled haploid plant by HDR with the use of altered CENH3 and a Pol theta knock-out mutation (or additional a knock-out of KU70, KU80, LiglV or any possible combination). Parent plant 1 has haploid inducer activity based on an altered CENH3 gene, a knock-out mutation in PolQ alone or combined with a knock-out of KU70, KU80, LiglV or any possible combination and also carries the GE components (SEQ ID NO: 86, 87, 90 and 100) together with a repair template for HDR (SEQ ID NO: 101 ), integrated via transformation as described above. The GE components are directed to an ALS gene intruding a S653N mutation which results in increased herbicide resistance (Wolters et al. (2018), Efficient in plants gene targeting in Arabidopsis using egg cell-specific expression of the Cas9 nuclease of Staph- yloccus aureus, The Plant Journal, 94, 735,746). After crossing with parent plant 2, the GE component edits the genome of parent plant 2 in the early zygote. The mutation in PolQ or the combination with mutations of any NHEJ-factor mediates strong reduction of NHEJ and drives HDR. The genome of parent plant 1 including all transgenic elements is completely removed. The haploid plant with the gene editing event is analyzed for increased herbicide resistance and doubled to generate a homogenous edited doubled haploid plant.

Example 11 : Generation of homogenous GE edited doubled haploid plants by HDR with the use of a Pol theta knock-out mutation and a knock-out of KU70 or KU80

Pol theta and Ku70 or Ku80 knock out mutants are generated as described above.

Figure 4 shows a schematic overview for the generation of a GE edited doubled haploid plant by HDR with the use of a Pol theta knock-out mutation and a knock-out of KU70 or KU80. Parent plant 1 has haploid inducer activity based on a KU70 or KU80 endogenous gene knock-out, it also carries a knock-out mutation in PolQ and the GE components (SEQ ID NO: 86, 87, 90 and 100) together with a repair template for HDR targeting the ALS gene (SEQ ID NO: 101 ), integrated via transformation as described above. After crossing with parent plant 2, the GE component edits the genome of parent plant 2 in the early zygote. The mutation in PolQ and the mutation in KU70 or KU80 mediates strong reduction of NHEJ and drives HDR. The genome of parent plant 1 including all transgenic elements is completely removed. The haploid plant with the gene editing event is analyzed for increased herbicide resistance and doubled to generate a homogenous edited doubled haploid plant.

Example 12: Generation of homogenous GE edited (SDN 1 or SDN2) doubled haploid plants by using altered CENH3 and co-edited endogenous gene as selection marker or identifiable phenotype.

Altered CENH3 mutants are generated as described above.

Figure 5 shows a schematic overview for the generation of a GE edited doubled haploid plant by using altered CENH3 and co-edited endogenous gene as selection marker or iden- tifiable phenotype. Parent plant 1 has haploid inducer activity based on an altered CENH3 (generated as described above) and carries a GE plasmid (SEQ ID NO: 86), integrated via transformation as described above. After crossing with parent plant 2, the GE component edits the genome of parent plant 2 in the early zygote. In addition to the edit in the target gene (either Adh1 as described above or any other target gene), a co-edit in a selectable marker (here TT4, or any other selectable or visible marker) is made. The genome of parent plant 1 including all transgenic elements is completely removed. The haploid plant with the SDN1 event is identified based on the co-edited endogenous gene to assist with identification or selection, further analyzed and doubled to generate a homogenous edited doubled haploid plant. Example 13: Generation of homogenous GE edited (SDN1 or SDN2) doubled haploid plants by using KU70 or KU80 knock-out mutants (or any other of the above mentioned proteins conferring haploid inducer activity) and co-edited endogenous gene as selection marker or identifiable phenotype.

KU70 or KU80 knock out mutants are generated as described above.

Figure 6 shows a schematic overview for the generation of a GE edited doubled haploid plant by using KU70 or KU80 knock-out mutants and co-edited endogenous gene as selection marker or identifiable phenotype. Parent plant 1 has haploid inducer activity based on a KU70 or KU80 endogenous gene knock-out and carries a GE plasmid (SEQ ID NO: 86), integrated via transformation as described above. After crossing with parent plant 2, the GE component edits the genome of parent plant 2 in the early zygote. In addition to the edit in the target gene (either Adh1 as described above or any other target gene), a co-edit in a selectable marker (here TT4, or any other selectable or visible marker) is made. The genome of parent plant 1 including all transgenic elements is completely removed. The haploid plant with the SDN1 event is identified based on the selectable marker, further analyzed and doubled to generate a homogenous edited doubled haploid plant.

Example 14: Generation of homogenous GE edited doubled haploid plants by HDR with use of an altered CENH3 and a Pol theta knock-out (or additional a knock-out of KU70, or KU80, or LiglV, or any possible combination) and co-edited endogenous gene as selection marker or identifiable phenotype. The mutants are generated as described above.

Figure 7 shows a schematic overview for the generation of a GE edited doubled haploid plant by HDR with use of an altered CENH3 and a Pol theta knock-out (or additional a knock-out of KU70, or KU80, or LiglV, or any possible combination) and co-edited endogenous gene as selection marker or identifiable phenotype. Parent plant 1 has haploid in- ducer activity based on altered CENH3 and carries a Pol theta knock-out mutation (alone or in combination with a knock-out mutation in KU70, KU80, or LiglV, or any combination thereof) and GE components (SEQ ID NO: 86, 87, 90 and 100) together with a repair template (SEQ ID NO: 101 ) targeting the ALS gene, integrated via transformation as described above. After crossing with parent plant 2, the GE component edits the genome of parent plant 2 in the early zygote. The mutation in PolQ or combination of PolQ and any NHEJ- factor mutations mediates strong reduction of NHEJ and drives HDR. In addition to the edit in the target gene (either Adh1 as described above or any other target gene), a co-edit in a selectable marker (here TT4, or any other selectable or visible marker) is made. The genome of parent plant 1 including all transgenic elements is completely removed. The haploid plant with the gene editing event is identified based on the selectable marker or identifiable phenotype, further analyzed for increased herbicide resistance and doubled to generate a homogenous edited doubled haploid plant.

Example 15: Generation of homogenous GE edited doubled haploid plants by HDR with use of a Pol theta and KU70 or Pol theta and KU80 knock-out and co-edited endogenous gene as selection marker or identifiable phenotype. Mutants are generated as described above.

Figure 8 shows a schematic overview for the generation of a GE edited doubled haploid plant by HDR with use of a Pol theta and KU70 or Pol theta and KU80 knock-out and coedited endogenous gene as selection marker or identifiable phenotype. Parent plant 1 has haploid inducer activity based on a knock-out mutation in KU70 or KU80, and carries knockout mutation in PolQ and GE components (SEQ ID NO: 86, 87, 90 and 100) together with a repair template (SEQ ID NO: 101 ), integrated via transformation as described above. After crossing with parent plant 2, the GE component edits the genome of parent plant 2 in the early zygote. The mutation in PolQ or combination of PolQ and any NHEJ-factor muta- tions mediates strong reduction of NHEJ and drives HDR. In addition to the edit in the target gene (either Adh1 as described above or any other target gene), a co-edit in a selectable marker (here TT4, or any other selectable or visible marker) is made. The genome of parent plant 1 including all transgenic elements is completely removed. The haploid plant with the gene editing event is identified based on the selectable marker, further analyzed for in- creased herbicide resistance and doubled to generate a homogenous edited doubled haploid plant.

Claims

1. A method for generating a haploid plant cell comprising at least one modification at one or more predetermined genetic location(s), wherein the method comprises the following steps: (a) providing a first gamete cell of a cellular system having biological activity of a haploid inducer, wherein the first gamete cell comprises

(II) at least one introduced site-specific nuclease, or a sequence encoding the same, the site-specific nuclease induces at least one single-strand break or at least one double-strand break at one or more predetermined locations) in the genome of a second gamete cell of a target cellular system before the first cell division or at least during the first and second cell division after gamete fusion; and optionally: at least one nucleic acid sequence of interest flanked by one or more homology sequence(s) complementary to one or more nucleic acid sequence(s) adjacent to one or more predetermined location(s) in the genetic material of the second gamete cell of the target cellular system; and

(b) allowing the first gamete cell to fuse with a second gamete cell of a target cellular system to generate a zygote cell, the second gamete cell comprises the one or more predetermined genetic location(s).

2. The method according to claim 1 , further comprising the steps: (c) culturing the zygote cell such that

(i) the at least one site-specific nuclease and optionally the at least one nucleic acid of interest is expressed in the zygote cell to induce the at least one modification at the one or more predetermined location(s) in the genome of the second gamete cell before the first cell division or at least during the first and second cell division after gamete fusion, wherein the at least one modification is effected by DNA repair, preferably through homology-directed repair using the at least one nucleic acid of interest as a repair template; and

(ii) the set of chromosomes of the first gamete cell is eliminated; and (d) obtaining a haploid cell comprising the genome of the second gamete cell of the target cellular system including the at least one modification at the one or more predetermined genetic location(s).

3. The method according to claim 1 or 2, wherein the at least one site-specific nuclease and optionally the at least one nucleic acid of interest is operably linked to a promoter, which is active in the early zygote, preferably before the first cell division, or at least during the first and second cell division after gamete fusion.

4. The method according to claim 3, wherein the promoter is selected from the group consisting of (p)BdUbM O promoter (SEQ ID NO: 1 ), a (p)ZmUbM promoter (SEQ ID NO: 2), a (p)OsActin promoter (SEQ ID NO: 3), and a single or double 35S promoter (SEQ ID NO: 4), optionally including an ZmUbM intron, an BdUbM 0 intron and/or an Adh1 intron, (SEQ ID NOs: 5 to 1 1 ), or any combination thereof, or a sequence having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity when compared over the whole length of the respective sequence of any one of SEQ ID NOs: 1 to 1 1.

5. The method according to any preceding claim, wherein the first gamete cell of the cellular system having biological activity of a haploid inducer comprises a polynucleotide which comprises a nucleotide sequence encoding for a protein, wherein the polynucleotide comprises at least one mutation causing an alteration of the amino acid sequence of the protein and said alteration confers the biological activity of a haploid inducer, wherein the protein is selected from the group consisting of: CENH3, Ku70, Ku80, KNL2, Cenp-C, PLA1 and PLA1 combined with a myosin heavy chain protein, preferably Ku70 and/or Ku80.

6. The method according to any preceding claim, wherein the at least one nucleic acid sequence of interest introduced into the first gamete cell of the cellular system having biological activity of a haploid inducer is selected from the group consisting of: an exoge- nous gene, a modified endogenous gene, a synthetic sequence, an intronic sequence, a coding sequence or a regulatory sequence.

7. The method according to any preceding claim, wherein the at least one modification at one or more predetermined genetic location(s) in the genome of the second gamete cell results in the introduction of a trait of interest into the genetic material of the haploid cell.

8. The method according to claim 7, wherein the at least one modification at one or more predetermined genetic location(s) in the genome of the second gamete cell further results in the introduction of a selection marker into the genetic material of the haploid cell.

9. The method according to any preceding claim, wherein the at least one modification is selected from a replacement of at least one nucleotide; a deletion of at least one nucleotide; an insertion of at least one nucleotide or any combination thereof.

10. The method according to any preceding claim, wherein the method further comprises the step of culturing the haploid cell under conditions to obtain a haploid cellular system comprising the genome of the second gamete cell of the target cellular system including the at least one modification at the one or more predetermined genetic location(s), preferably wherein the haploid cellular system is a plant.

11. The method according to any preceding claim, wherein the cellular system having biological activity of a haploid inducer is a plant and/or the target cellular system is a plant.

12. A method for generating a doubled haploid cell or a doubled haploid cellular system comprising at least one modification at one or more predetermined genetic locations), wherein the method comprises (a) generating a haploid cell comprising at least one modification at one or more predetermined genetic location(s) by the method of claims 1-11 ;

13. A haploid cellular system obtained by a method according to claims 10 or 1 1.

14. A doubled haploid cellular system obtained by a method according claim 12.

15. A cellular system having biological activity of a haploid inducer, comprising

(a) an inactivated or partially inactivated Polymerase theta enzyme, or a sequence encoding the same;

(c) at least one site-specific nuclease, or a sequence encoding the same, the site-specific nuclease induces a double-strand break at one or more predetermined location(s) in the genome of a target cellular system; optionally: at least one nucleic acid of interest flanked by one or more homology sequence(s) complementary to one or more nucleic acid sequence(s) adjacent to one or more predetermined location(s) in the genetic material of a target cellular system, preferably wherein the cellular system having biological activity of a haploid inducer and the target cellular system is a plant.

16. Use of a cellular system having biological activity of a haploid inducer according to claim 15 for generating a haploid cellular system comprising at least one modification at one or more predetermined genetic location(s) or for generating a doubled haploid cellular system comprising at least one modification at one or more predetermined genetic locations).