CN116042573A

CN116042573A - Method for improving base editing efficiency of guide editing system

Info

Publication number: CN116042573A
Application number: CN202211488466.1A
Authority: CN
Inventors: 杨进孝; 徐雯; 赵久然; 杨永星; 张璐
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2022-11-25
Filing date: 2022-11-25
Publication date: 2023-05-02

Abstract

The invention discloses a method for improving the base editing efficiency of a guide editing system. In order to further improve the editing efficiency of the guide editing system, the invention knocks out the rice MMR repair gene on the basis of the PE-P6 guide editing system to obtain the guide editing system PE-P6 ^ΔOsMLH1 RT-S and guidance editing system PE-P6 ^ΔOsMLH1 RT-M. With PECompared with the P6 guided editing system, after the OsMLH1 gene is knocked out, the guided editing system PE-P6 ^ΔOsMLH1 RT-S and PE-P6 ^ΔOsMLH1 The RT-M can further improve the editing efficiency of the target point; and boot editing system PE-P6 ^ΔOsMLH1 The RT-M can improve the editing efficiency of the rice target point to the greatest extent.

Description

Method for improving base editing efficiency of guide editing system

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for improving the base editing efficiency of a guide editing system.

Background

In 2019, a multifunctional genome editing technology, i.e., guided editing (PE), was newly developed by David Liu laboratories, which can precisely implement 12 types of base substitutions, small insertions, and deletions without double-stranded DNA breaks or donor DNA templates. The minimal composition of the PE system is a Cas9 nickase (Cas 9 nickase, cas9 n) and Moloney mouse leukemia virus reverse transcriptase (M-MLV reverse transcriptase, M-MLV RT), and a guide RNA (prime editing guide RNA, pegRNA) that directs editing. The pegRNA comprises a spacer region specifying the target site, a single-guide RNA (sgRNA) scaffold and a 3' extension. Wherein the 3' extension comprises a primer binding site (primer binding site, PBS) complementary to a portion of the DNA spacer region, and an RT template encoding the desired editing and downstream genomic sequence. 2021, an engineered pegRNA (epegRNA) integrates a structured RNA motif into the 3' end of the pegRNA, improving the efficiency of guided editing in human cells by 3-4 fold.

The PE editing efficiency of most target sites in plants is much lower than 20% -70% in human cells. In recent years, a number of efforts including mainly optimizing PE components have been used to further enhance the application of guided editing in crops. For example, the use of polycistronic tRNA and ribozyme to promote expression of pegRNA in maize, the introduction of RT templates in conjunction with N-terminal reverse transcriptase-cas 9N fusion proteins for polynucleotide replacement in rice, and the removal of the ribonuclease H domain of M-MLV RT and the addition of viral nucleocapsid proteins with chaperone activity in rice and wheat can increase PE flexibility and applicability. However, after the above improvements, the average efficiency of PE in stable transgenic plants remains low. How to increase the efficiency of PE remains challenging, especially at the level of stable transformation of crops.

Disclosure of Invention

It is an object of the present invention to provide a kit.

The kit comprises a fusion protein, esgRNA and pegRNA;

the fusion protein sequentially comprises reverse transcriptase, cas9 nicking enzyme, self-cleaving oligopeptide and screening marker protein;

the esgRNA targets an MLH1 gene target sequence;

The pegRNA sequentially comprises esgRNA', a reverse transcription template sequence (RT sequence), a primer binding site sequence (PBS sequence), a connecting sequence and a tevopreQ1 motif; the esgRNA' targets the target sequence of the target gene.

In the complete system, the esgRNA sequentially comprises an MLH1 gene target sequence and an esgRNA skeleton.

Further, the esgRNA is an esgRNA with tRNA, and the tRNA is an RNA molecule obtained by replacing T in positions 1-77 of the sequence 12 with U. The esgRNA skeleton is an RNA molecule obtained by replacing T in 11368-11453 th site of a sequence 7 with U.

The number of esgrnas may be 1 or 2 or more.

The MLH1 gene sequence is shown in SEQ ID No. 13.

Further, 2 esgrnas are designated as esgRNA1 and esgRNA2, respectively, the target sequence in esgRNA1 is shown in positions 78-97 of sequence 12, and the target sequence in esgRNA2 is shown in positions 261-280 of sequence 12.

In the complete system, the pegRNA sequentially comprises a target sequence of a target gene, an esgRNA skeleton, an RT sequence, a PBS sequence, a connecting sequence and a tevopreQ1 motif.

The esgRNA skeleton is an RNA molecule obtained by replacing T in 11368-11453 th site of a sequence 7 with U.

The RT sequence is the reverse complementary sequence of 3 bases at the 3' -end of the target sequence and a section of genome sequence which is continuous behind the target sequence, and target mutation is introduced into the RT sequence, and is used as a reverse transcription template of reverse transcriptase, cDNA is reversely transcribed, and then the RT sequence is used as a repair template to repair genome DNA. The RT sequence can further be 8-34bp in size.

The PBS sequence (primer binding site sequence) is the reverse complementary sequence (n <17 > which is 1-n) of the target sequence from the nth base to the 17 th base of the 5' end of the target sequence.

The design methods or principles of the RT sequences and the PBS sequences can be referred to as those reported in the prior art for the design methods or principles related to the RT sequences and the PBS sequences of pegRNA in the guidance editing technique (PE).

The RT sequence may be in the form of an RT-S template or an RT-M template, preferably an RT-M template. The RT sequence of the RT-S template form only comprises one target mutation site (i.e. the site where mutation is expected to occur), and mutation bases are introduced at the target mutation site. The RT sequence in the RT-M template form can introduce mutation bases at other sites (marked as additional mutation sites) except the target mutation site, namely, the introduced mutation bases in the RT sequence can be introduced at the target mutation site with no less than two mutation bases, or can be introduced at the target mutation site with additional mutation bases at other sites except the target mutation site. The additional mutation site may be any site in the RT sequence other than the mutation site of interest. In practical application, a proper site can be selected as an additional mutation site according to practical needs, and a proper mutation base is introduced, for example, when only the base of the target mutation site is expected to be mutated to cause the change of the amino acid sequence, and the base of the additional mutation site is not caused to cause the change of the amino acid sequence after the mutation, synonymous mutation can be carried out on the base of the additional mutation site through design. Further, the way of introducing the mutant base is base substitution. Further, the number of mutant bases introduced at the target mutation site may be one or two or more. In an embodiment of the present invention, the number of mutation bases introduced at the target mutation site is specifically one. The number of additional mutation bases introduced at other sites than the target mutation site may be one or two or more.

The linking sequence may be of any length. In a specific embodiment of the invention, the linker sequence is a 8bp linker sequence and is designated as an 8-bp linker, which can be designed by pegLIT (https:// doi. Org/10.1038/s 41587-021-01039-7) according to methods known in the art. In a specific embodiment of the invention, the 8-bp linker is aacgagag, taaatatt, aaagaaga, atataatc, actctctg, cgaagagg, aagataac, aagtctta, ttataaga, aaggaagg or aattataa.

The nucleotide sequence of the tevopreQ1 motif is shown in sequence 8.

In the complete system, the pegRNA is driven to express by a composite promoter; the composite promoter comprises an E35S promoter, a CmYLCV promoter and a OsU promoter in sequence.

In a specific embodiment of the invention, the composite promoter consists of an E35S promoter, a CmYLCV promoter and a OsU3 promoter in sequence.

The nucleotide sequence of the E35S promoter is shown in 10025-10461 of the sequence 7.

The nucleotide sequence of the CmYLCV promoter is shown in positions 10462-10920 of the sequence 7.

The nucleotide sequence of the OsU promoter is shown in 10932-11270 of the sequence 7.

In the above-described kit, the Cas9 nickase may be various Cas9n or variants thereof known in the art, including bacterial-derived Cas9n (e.g., spCas9n, saCas9n-KKH, etc.), spCas9 variant nickases (e.g., xCas9n, cas9n-NG, cas9n-VQR, cas9n-VRER, etc.), cas9 high-fidelity enzyme variant nickases (e.g., hypas 9n, eSpCas9 (1.1) n, cas9-HF1n, etc.), etc.

Further, the Cas9 nickase is Cas9maxn; the Cas9maxn is A1) or A2):

a1 Amino acid sequence is a protein shown in sequence 5;

a2 A protein having the same function and obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 5.

Still further, the Cas9maxn is a 1) or a 2) or a 3):

a1 A cDNA molecule or a DNA molecule shown in positions 4422-8522 of SEQ ID NO. 7;

a2 A cDNA molecule or DNA molecule having 75% or more identity to the nucleotide sequence defined in a 1) and encoding the Cas9maxn;

a3 Under stringent conditions with the nucleotide sequence defined in b 1) or b 2), and a cDNA molecule or DNA molecule encoding the Cas9 maxn.

The reverse transcriptase may be a reverse transcriptase derived from a virus such as Moloney mouse leukemia virus (Moloney murine leukemia virus, M-MLV), a reverse transcriptase derived from cauliflower mosaic virus (CaMV), etc., or a virus derived from a bacterium such as Escherichia coli.

Further, the reverse transcriptase is M-MLV RT; the M-MLV RT is B1) or B2):

b1 Amino acid sequence is a protein shown in sequence 1;

b2 A protein having the same function and obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 1.

Still further, the coding gene of the M-MLV RT is b 1) or b 2) or b 3):

b1 A cDNA molecule or a DNA molecule shown in positions 2292-4322 of SEQ ID NO. 7;

b2 A cDNA molecule or DNA molecule having 75% or more identity to the nucleotide sequence defined in b 1) and encoding said M-MLV RT;

b3 Under stringent conditions with the nucleotide sequence defined under b 1) or b 2) and a cDNA molecule or DNA molecule encoding said M-MLV RT.

The self-cleaving oligopeptide may be a 2A self-cleaving oligopeptide derived from a viral genome, such as a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine a rhinitis virus (ERAV) (E2A) peptide, a colletotrichum glomeroclade beta tetrad virus (Thosea asigna virus) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a taylor virus 2A peptide, and an encephalomyocarditis virus 2A peptide.

Further, the 2A self-cleaving oligopeptide from the viral genome is a 2A self-cleaving oligopeptide from porcine teschovirus-1; the amino acid sequence of the 2A self-cleaving oligopeptide (P2A) from porcine teschovirus-1 is C1) or C2):

C1 Amino acid sequence is a protein shown in sequence 3;

c2 A protein having the same function and obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 3.

The coding gene of the P2A is c 1) or c 2) or c 3):

c1 A cDNA molecule or a DNA molecule shown in positions 8673-8729 of SEQ ID NO. 7;

c2 A cDNA molecule or DNA molecule having 75% or more identity to the nucleotide sequence defined in c 1) and encoding said P2A;

c3 Under stringent conditions with the nucleotide sequence defined under c 1) or c 2), and a cDNA molecule or DNA molecule encoding said P2A.

The screening agent resistance protein may be various resistance proteins known in the art, such as kanamycin resistance protein, herbicide resistance proteins (e.g., glyphosate resistance protein, glufosinate resistance protein), phosphomannose isomerase, and the like.

Further, the screening agent resistance protein is hygromycin phosphotransferase; the hygromycin phosphotransferase is D1) or D2):

d1 Amino acid sequence is a protein shown in sequence 4;

d2 A protein having the same function and obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 4.

Still further, the coding gene of hygromycin phosphotransferase is d 1) or d 2) or d 3):

d1 A cDNA molecule or a DNA molecule shown at positions 8730-9755 of SEQ ID NO. 7;

d2 A cDNA molecule or DNA molecule having 75% or more identity to the nucleotide sequence defined in d 1) and encoding said hygromycin phosphotransferase;

d3 Under stringent conditions with the nucleotide sequence defined in d 1) or d 2), and a cDNA molecule or DNA molecule encoding said hygromycin phosphotransferase.

It is another object of the present invention to provide a new use of the above-described kit.

The invention provides the use of the above described kit in any one of the following S1) to S4):

s1) editing genome sequences of organisms or biological cells;

s2) preparing an edited product of genomic sequences of an organism or a biological cell;

s3) improving the editing efficiency of genome sequences of organisms or biological cells;

s4) preparing a product for improving the editing efficiency of genome sequences of organisms or biological cells.

It is a further object of the invention to provide a method as described in any one of the following T1) -T3):

t1) a method for editing a genomic sequence, comprising the steps of: allowing an organism or biological cell to express the fusion protein, the esgRNA and the pegRNA;

T2) a method for increasing the efficiency of editing genomic sequences of an organism or a biological cell, comprising the steps of: allowing an organism or biological cell to express the fusion protein, the esgRNA and the pegRNA;

t3) a method for preparing a biological mutant, comprising the following steps: editing the genome sequence of the organism or the biological cell according to the method described in T1) or T2) to obtain the biological mutant.

In the above methods, in the above T1) and T2), the method of expressing the above fusion protein, the above esgRNA and the above pegRNA in an organism or a biological cell is a method of introducing a gene encoding the above fusion protein, a DNA molecule transcribed the above esgRNA and a DNA molecule transcribed the above pegRNA into a plant of interest.

Further, in the T1) and T2), the gene encoding the fusion protein, the DNA molecule transcribed to the esgRNA, and the DNA molecule transcribed to the pegRNA are introduced into a plant of interest through a recombinant expression vector. The coding gene of the fusion protein, the DNA molecule for transcribing the esgRNA and the DNA molecule for transcribing the pegRNA can be introduced into a target plant through the same recombinant expression vector, or can be jointly introduced into the target plant through two or more recombinant expression vectors.

In a specific embodiment of the invention, the gene encoding the fusion protein, the DNA molecule transcribed into the above esgRNA and the DNA molecule transcribed into the above pegRNA are introduced into the plant of interest via the same recombinant expression vector. The recombinant expression vector comprises an expression cassette which sequentially consists of a promoter, an encoding gene of M-MLV RT, an encoding gene of Cas9maxn, an encoding gene of self-cleaving oligopeptide P2A, an encoding gene of screening agent resistance protein HPT and a terminator, an expression cassette which sequentially consists of the promoter, a DNA molecule for transcribing esgRNA and poly T, and an expression cassette which sequentially consists of the promoter, the DNA molecule for transcribing pegRNA and poly T. The recombinant expression vector is in particular PE-P6 as follows ^ΔOsMLH1 RT-S-1 vector, PE-P6 ^ΔOsMLH1 RT-S-2 vector, PE-P6 ^ΔOsMLH1 RT-S-3 vector, PE-P6 ^ΔOsMLH1 RT-M-1 vector, PE-P6 ^ΔOsMLH1 RT-M-2 vector or PE-P6 ^ΔOsMLH1 RT-M-3 vector.

In any of the above kits or applications or methods, the editing of the genomic sequence includes base substitution (e.g., single base substitution and multiple base substitution), base insertion (e.g., single base insertion and multiple base insertion), and base deletion (e.g., single base deletion and multiple base deletion) of the genomic sequence. In a specific embodiment of the invention, the editing of the genomic sequence is a base substitution of the genomic sequence.

In any of the above kits or uses or methods, the organism is X1) or X2) or X3) or X4):

x1) plants or animals;

x2) monocotyledonous or dicotyledonous plants;

x3) a gramineous plant;

x4) rice.

The biological cell is Y1) or Y2) or Y3) or Y4):

y1) plant cells or animal cells;

y2) monocot or dicot cells;

y3) a graminaceous plant cell;

y4) rice cells.

In order to further improve the editing efficiency of the guide editing system, the invention knocks out the rice MMR repair gene on the basis of the PE-P6 guide editing system to obtain the guide editing system PE-P6 ^ΔOsMLH1 RT-S and guidance editing system PE-P6 ^ΔOsMLH1 RT-M. Compared with PE-P6 guided editing system, after the OsMLH1 gene is knocked out, the guided editing system PE-P6 ^ΔOsMLH1 RT-S and PE-P6 ^ΔOsMLH1 The RT-M can further improve the editing efficiency of the target point; and boot editing system PE-P6 ^ΔOsMLH1 The RT-M can improve the editing efficiency of the rice target point to the greatest extent.

Drawings

FIG. 1 shows a boot editing system PE-P3, PE-P4, PE-P5, PE-P6, PE-P7, PE-P6 ^OE ^hMLHdn RT-S、PE-P6 ^OE ^hMLHdn RT-M、PE-P6 ^ΔOsMLH1 RT-S and PE-P6 ^ΔOsMLH1 Schematic structural diagram of RT-M expression vector.

FIG. 2 is a schematic representation of the form of an RT-M template.

FIG. 3 shows the efficiency of rice callus editing for 8 targets in the guidance editing systems PE-P3, PE-P4, PE-P5, PE-P6, PE-P7.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

In the following examples, the editing efficiency of the guidance editing system in rice calli = (number of reads with all mutation sites/total number of reads detected by group 1 ×100% + number of reads with all mutation sites detected by group 2 ×100% + number of reads with all mutation sites detected by group 3/total number of reads ×100%)/4.

Editing efficiency of the pilot editing system in rice T0 seedlings = number of positive T0 seedlings mutated at all mutation sites/total number of positive T0 seedlings analyzed x 100%.

Paddy rice in Nippon sunny days: reference is made to: liang Weigong, wang Gaohua, du Jingyao, et al sodium nitroprusside and its photolysis products have an effect on the growth of young seedlings of Nippon rice and the expression of 5 hormone marker genes [ J ]. University of Henan university (Nature edition), 2017 (2): 48-52; the public is available from the academy of agriculture and forestry, beijing, city.

Recovery medium: n6 solid medium containing 200mg/L of timentin.

Screening the culture medium: n6 solid medium containing 50mg/L hygromycin.

Differentiation medium: n6 solid medium containing 2mg/L KT, 0.2mg/L NAA, 0.5g/L glutamic acid, 0.5g/L proline.

Rooting medium: n6 solid medium containing 0.2mg/L NAA, 0.5g/L glutamic acid, 0.5g/L proline.

Example 1 design of the Main elements of different guide editing systems and design of expression vector thereof

1. Principal element design for different guided editing systems

The guidance editing system PE-P3 comprises fusion proteins, esgRNA and pegRNA; fusion proteins include Cas9 nicking enzymes (e.g., cas9n (H840A)), reverse transcriptases (e.g., M-MLV), self-cleaving oligopeptides (e.g., P2A), and selectable marker proteins (e.g., HPT); esgRNA is used to create non-editing strand breaks; the pegRNA consists of esgRNA, reverse transcription template sequence (RT sequence) and primer binding site sequence (PBS sequence) in sequence.

The guide editing system PE-P4 comprises fusion protein and pegRNA; fusion proteins include Cas9 nicking enzymes (e.g., cas9n (H840A)), reverse transcriptases (e.g., M-MLV), self-cleaving oligopeptides (e.g., P2A), and selectable marker proteins (e.g., HPT); the pegRNA consists of esgRNA, reverse transcription template sequence (RT sequence) and primer binding site sequence (PBS sequence) in sequence.

The guide editing system PE-P5 comprises fusion protein and pegRNA; fusion proteins include Cas9 nicking enzymes (e.g., cas9n (Cas 9 maxn) with increased cleavage efficiency for R221K, N394K mutations), reverse transcriptases (e.g., M-MLV), self-cleaving oligopeptides (e.g., P2A), and selectable marker proteins (e.g., HPT); the pegRNA consists of esgRNA, a reverse transcription template sequence (RT sequence) and a primer binding site sequence (PBS sequence) in sequence, and a composite promoter system (E35S+CmYLCV+ OsU 3) drives the expression of the pegRNA.

The guide editing system PE-P6 comprises fusion protein and pegRNA; fusion proteins include Cas9 nicking enzymes (e.g., cas9n (Cas 9 maxn) with increased cleavage efficiency for R221K, N394K mutations), reverse transcriptases (e.g., M-MLV), self-cleaving oligopeptides (e.g., P2A), and selectable marker proteins (e.g., HPT); the pegRNA consists of esgRNA, reverse transcription template sequence (RT sequence) and primer binding site sequence (PBS sequence) in sequence, and 8-bp linker and tevopreQ1 motif are introduced at the 3 'end of the pegRNA (to form an engineered pegRNA (epegRNA) to further enhance the stability of the 3' end of the pegRNA from degradation), and a composite promoter system (E35S+CmYLCV+ OsU 3) drives pegRNA expression.

The guide editing system PE-P7 comprises fusion protein and pegRNA; fusion proteins include Cas9 nicking enzymes (e.g., cas9n (Cas 9 maxn) with increased cleavage efficiency for R221K, N394K mutations), reverse transcriptases (e.g., M-MLV), self-cleaving oligopeptides (e.g., P2A), and selectable marker proteins (e.g., HPT); the pegRNA consists of esgRNA, reverse transcription template sequence (RT sequence) and primer binding site sequence (PBS sequence) in sequence, and 8-bp linker and mpknot motif are introduced at the 3 'end of the pegRNA (to form an engineered pegRNA (epegRNA) to further enhance the stability of the 3' end of the pegRNA from degradation), and a composite promoter system (E35S+CmYLCV+ OsU 3) drives pegRNA expression.

Guide editing system PE-P6 ^OE ^hMLH1dn RT-S includes fusion proteins, hMLH1dn and pegRNA; fusion proteins include Cas9 nicking enzymes (e.g., cas9n (Cas 9 maxn) with increased cleavage efficiency for R221K, N394K mutations), reverse transcriptases (e.g., M-MLV), self-cleaving oligopeptides (e.g., P2A), and selectable marker proteins (e.g., HPT); the pegRNA sequentially comprises esgRNA, a reverse transcription template sequence (RT sequence) and a primer binding site sequence (PBS sequence), wherein the RT sequence is in the form of an RT-S template (only mutation bases are introduced into target mutation sites, and all mutation sites are only of interest)Target mutation site) and 8-bp linker and tevopreQ1 motifs were introduced at the 3 'end of the pegRNA (to form an engineered pegRNA (epegRNA) to further enhance stability of the 3' end of the pegRNA against degradation), the composite promoter system (e35s+cmylcv+ OsU 3) driven pegRNA expression.

Guide editing system PE-P6 ^OE ^hMLH1dn RT-M includes fusion proteins, hMLH1dn and pegRNA; fusion proteins include Cas9 nicking enzymes (e.g., cas9n (Cas 9 maxn) with increased cleavage efficiency for R221K, N394K mutations), reverse transcriptases (e.g., M-MLV), self-cleaving oligopeptides (e.g., P2A), and selectable marker proteins (e.g., HPT); the pegRNA consists of esgRNA, a reverse transcription template sequence (RT sequence) and a primer binding site sequence (PBS sequence) in sequence, wherein the RT sequence is in the form of an RT-M template (a mutation base is introduced at a target mutation site, an additional mutation base is introduced at other sites (additional mutation sites) besides the target mutation site, all mutation sites consist of the target mutation site and the additional mutation site), an 8-bp linker and a tevopreQ1 motif are introduced at the 3 'end of the pegRNA (to form an engineered pegRNA (epegRNA) for further enhancing the stability of the 3' end of the pegRNA and preventing the pegRNA from being degraded), and a composite promoter system (E35S+CmYLCV+ OsU 3) drives the expression of the pegRNA.

Guide editing system PE-P6 ^ΔOsMLH1 RT-S includes fusion proteins, esgRNA and pegRNA; fusion proteins include Cas9 nicking enzymes (e.g., cas9n (Cas 9 maxn) with increased cleavage efficiency for R221K, N394K mutations), reverse transcriptases (e.g., M-MLV), self-cleaving oligopeptides (e.g., P2A), and selectable marker proteins (e.g., HPT); the esgRNA contains a target spot (the rice MMR repair gene is knocked out to prevent the intracellular MMR repair) for knocking out the rice MLH1 gene (the MLH1 gene sequence is shown as a sequence 14); the pegRNA sequentially comprises esgRNA, a reverse transcription template sequence (RT sequence) and a primer binding site sequence (PBS sequence), wherein the RT sequence is in the form of an RT-S template (only a mutation base is introduced at a target mutation site, all mutation sites are only target mutation sites), an 8-bp linker and a tevopreQ1 motif are introduced at the 3 '-end of the pegRNA (to form an engineered pegRNA (epegRNA) for further enhancing the stability of the 3' -end of the pegRNA and preventing the pegRNA from being degraded), and a composite promoter system (E35S+CmYLCV+ OsU 3) drives the expression of the pegRNA.

Guide editing system PE-P6 ^ΔOsMLH1 RT-M includes fusion proteins, esgRNA and pegRNA; fusion proteins include Cas9 nicking enzymes (e.g., cas9n (Cas 9 maxn) with increased cleavage efficiency for R221K, N394K mutations), reverse transcriptases (e.g., M-MLV), self-cleaving oligopeptides (e.g., P2A), and selectable marker proteins (e.g., HPT); the esgRNA contains a target point for knocking out rice MLH1 genes (knocking out rice MMR repair genes so as to prevent intracellular MMR repair); the pegRNA consists of esgRNA, a reverse transcription template sequence (RT sequence) and a primer binding site sequence (PBS sequence) in sequence, wherein the RT sequence is in the form of an RT-M template (a mutation base is introduced at a target mutation site, an additional mutation base is introduced at other sites (additional mutation sites) besides the target mutation site, all mutation sites consist of the target mutation site and the additional mutation site), an 8-bp linker and a tevopreQ1 motif are introduced at the 3 'end of the pegRNA (to form an engineered pegRNA (epegRNA) for further enhancing the stability of the 3' end of the pegRNA and preventing the pegRNA from being degraded), and a composite promoter system (E35S+CmYLCV+ OsU 3) drives the expression of the pegRNA.

The schematic diagram of RT-M in the epegRNA related to the guidance editing system is shown in FIG. 2, wherein red is the target mutation site, and blue is the additional mutation site.

The 8-bp linker in the epegRNA related to the guidance editing system is designed by pegLIT (https:// doi.org/10.1038/s 41587-021-01039-7).

2. Expression vector design for different guided editing systems

The schematic structure of the expression vector of each guidance editing system is shown in fig. 1.

Expression vectors that guide the editing system PE-P3 include M-MLV & Cas9n (H840A) & HPT expression cassettes, esgRNA expression cassettes, and pegRNA expression cassettes. In the M-MLV & Cas9N (H840A) & HPT expression cassette, M-MLV is fused at the N end of Cas9N (H840A), and simultaneously the screening agent resistance protein is fused at the C end of Cas9N (H840A) through self-cleaving polypeptide P2A, and the expression cassette is driven to be expressed by a ZmUbi1 promoter. The esgRNA expression cassette was driven by the OsU a promoter. The pegRNA expression cassette was driven by the OsU3 promoter.

Expression vectors guiding the editing system PE-P4 include M-MLV & Cas9n (H840A) & HPT expression cassettes and pegRNA expression cassettes. In the M-MLV & Cas9N (H840A) & HPT expression cassette, M-MLV is fused at the N end of Cas9N (H840A), and simultaneously the screening agent resistance protein is fused at the C end of Cas9N (H840A) through self-cleaving polypeptide P2A, and the expression cassette is driven to be expressed by a ZmUbi1 promoter. The pegRNA expression cassette was driven by the OsU3 promoter.

Expression vectors that guide the editing system PE-P5 include the M-MLV & Cas9maxn & HPT expression cassette and the pegRNA expression cassette. In the M-MLV & Cas9maxn & HPT expression cassette, M-MLV is fused at the N end of Cas9maxn, and simultaneously the screening agent resistance protein is fused at the C end of Cas9maxn through self-cleaving polypeptide P2A, and the expression cassette is driven to express by ZmUbi1 promoter. The pegRNA expression cassette was driven by the composite promoter E35S+CmYLCV+ OsU 3.

Expression vectors that guide the editing system PE-P6 include the M-MLV & Cas9maxn & HPT expression cassette and the pegRNA expression cassette. In the M-MLV & Cas9maxn & HPT expression cassette, M-MLV is fused at the N end of Cas9maxn, and simultaneously the screening agent resistance protein is fused at the C end of Cas9maxn through self-cleaving polypeptide P2A, and the expression cassette is driven to express by ZmUbi1 promoter. An 8-bp linker and tevopreQ1 motif are introduced into the 3' -end of the pegRNA expression cassette, and the expression cassette is driven to express by a composite promoter E35S+CmYLCV+ OsU 3.

Expression vectors that guide the editing system PE-P7 include the M-MLV & Cas9maxn & HPT expression cassette and the pegRNA expression cassette. In the M-MLV & Cas9maxn & HPT expression cassette, M-MLV is fused at the N end of Cas9maxn, and simultaneously the screening agent resistance protein is fused at the C end of Cas9maxn through self-cleaving polypeptide P2A, and the expression cassette is driven to express by ZmUbi1 promoter. 8-bp linker and tmpknot motif are introduced into the 3' end of pegRNA expression cassette, which is driven to express by the composite promoter E35S+CmYLCV+ OsU 3.

Guide editing system PE-P6 ^OE ^hMLH1dn Expression vectors for RT-S include M-MLV&Cas9maxn&HPT expression cassette, hMLH1dn expression cassette and pegRNA expression cassette. M-MLV&Cas9maxn&In the HPT expression cassette, M-MLV is fused at the N end of Cas9maxn, meanwhile, the screening agent resistance protein is fused at the C end of Cas9maxn through self-cleaving polypeptide P2A, and the expression cassette is driven to express by ZmUbi1 promoter. hMLH1dn expression cassette is started by OsActin The mover drives expression. The RT sequence in the pegRNA expression cassette is in the form of RT-S template, and 8-bp linker and tevopreQ1 motif are introduced into the 3' end of the pegRNA, and the expression cassette is driven to express by a composite promoter E35S+CmYLCV+ OsU 3.

Guide editing system PE-P6 ^OE ^hMLH1dn Expression vectors for RT-M include M-MLV&Cas9maxn&HPT expression cassette, hMLH1dn expression cassette and pegRNA expression cassette. M-MLV&Cas9maxn&In the HPT expression cassette, M-MLV is fused at the N end of Cas9maxn, meanwhile, the screening agent resistance protein is fused at the C end of Cas9maxn through self-cleaving polypeptide P2A, and the expression cassette is driven to express by ZmUbi1 promoter. The hMLH1dn expression cassette was driven by the osain promoter. The RT sequence in the pegRNA expression cassette is in the form of RT-M template, and 8-bp linker and tevopreQ1 motif are introduced into the 3' end of the pegRNA, and the expression cassette is driven to express by a composite promoter E35S+CmYLCV+ OsU 3.

Guide editing system PE-P6 ^ΔOsMLH1 Expression vectors for RT-S include M-MLV&Cas9maxn&HPT expression cassette, esgRNA expression cassette, and pegRNA expression cassette. M-MLV&Cas9maxn&In the HPT expression cassette, M-MLV is fused at the N end of Cas9maxn, meanwhile, the screening agent resistance protein is fused at the C end of Cas9maxn through self-cleaving polypeptide P2A, and the expression cassette is driven to express by ZmUbi1 promoter. The esgRNA expression cassette contains a target for knocking out the rice MLH1 gene (sequence 13), and the expression cassette is driven to express by OsU a. The RT sequence in the pegRNA expression cassette is in the form of RT-S template, and 8-bp linker and tevopreQ1 motif are introduced into the 3' end of the pegRNA, and the expression cassette is driven to express by a composite promoter E35S+CmYLCV+ OsU 3.

Guide editing system PE-P6 ^ΔOsMLH1 Expression vectors for RT-M include M-MLV&Cas9maxn&HPT expression cassette, esgRNA expression cassette, and pegRNA expression cassette. M-MLV&Cas9maxn&In the HPT expression cassette, M-MLV is fused at the N end of Cas9maxn, meanwhile, the screening agent resistance protein is fused at the C end of Cas9maxn through self-cleaving polypeptide P2A, and the expression cassette is driven to express by ZmUbi1 promoter. The esgRNA expression cassette contains a target for knocking out rice MLH1 gene, and the expression cassette is driven to express by OsU a. The RT sequence in the pegRNA expression cassette is in the form of RT-M template, and 8-bp linker and tevopreQ1 motif are introduced into the 3' -end of pegRNA, and the expression cassette is formed by a compound type promoterThe promoter E35S+CmYLCV+ OsU3 drives expression.

Example 2 construction of expression vectors of different guide editing systems and comparison of efficiency of base editing of Rice genome

1. Construction of expression vectors for different guidance editing systems

The following recombinant vectors were constructed artificially, each of which was a circular plasmid:

the total number of the expression vectors of the guiding editing system PE-P3 is 8, and the expression vectors are PE-P3-RTM-OsALS-3, PE-P3-RTM-OsEPSPS-2, PE-P3-RTM-OsGRF4-T2, PE-P3-RTM-OsSD1, PE-P3-RTM-OsCold1, PE-P3-RTM-OsALS-1, PE-P3-RTM-OsACC-1 and PE-P3-RTM-OsGS3 vectors respectively.

The total number of the expression vectors of the guide editing system PE-P4 is 8, and the expression vectors are PE-P4-RTM-OsALS-3, PE-P4-RTM-OsEPSPS-2, PE-P4-RTM-OsGRF4-T2, PE-P4-RTM-OsSD1, PE-P4-RTM-OsCold1, PE-P4-RTM-OsALS-1, PE-P4-RTM-OsACC-1 and PE-P4-RTM-OsGS3 respectively.

The total number of the expression vectors of the guiding editing system PE-P5 is 8, and the expression vectors are PE-P5-RTM-OsALS-3, PE-P5-RTM-OsEPSPS-2, PE-P5-RTM-OsGRF4-T2, PE-P5-RTM-OsSD1, PE-P5-RTM-OsCold1, PE-P5-RTM-OsALS-1, PE-P5-RTM-OsACC-1 and PE-P5-RTM-OsGS3 respectively.

The total number of the expression vectors of the guide editing system PE-P6 is 8, and the expression vectors are PE-P6-RTM-OsALS-3, PE-P6-RTM-OsEPSPS-2, PE-P6-RTM-OsGRF4-T2, PE-P6-RTM-OsSD1, PE-P6-RTM-OsCold1, PE-P6-RTM-OsALS-1, PE-P6-RTM-OsACC-1 and PE-P6-RTM-OsGS3 respectively.

The total number of the expression vectors of the guide editing system PE-P7 is 8, and the expression vectors are PE-P7-RTM-OsALS-3, PE-P7-RTM-OsEPSPS-2, PE-P7-RTM-OsGRF4-T2, PE-P7-RTM-OsSD1, PE-P7-RTM-OsCold1, PE-P7-RTM-OsALS-1, PE-P7-RTM-OsACC-1 and PE-P7-RTM-OsGS3 respectively.

Guide editing system PE-P6 ^OE ^hMLH1dn RT-S expression vectors total 3, PE-P6 respectively ^OE ^hMLHdn RT-S-OsEPSPS-2、PE-P6 ^OE ^hMLHdn RT-S-OsGRF4-T2、PE-P6 ^OE ^hMLHdn RT-S-OsSD1 vector.

Guide editing system PE-P6 ^OE ^hMLH1dn RT-M expression vectors total 3, PE-P6 respectively ^OE ^hMLHdn RT-M-OsEPSPS-2、PE-P6 ^OE ^hMLHdn RT-M-OsGRF4-T2、PE-P6 ^OE ^hMLHdn RT-M-OsSD1 vector.

Guide editing system PE-P6 ^ΔOsMLH1 RT-S expression vectors total 3, PE-P6 respectively ^ΔOsMLH1 RT-S-OsEPSPS-2、PE-P6 ^ΔOsMLH1 RT-S-OsGRF4-T2、PE-P6 ^ΔOsMLH1 RT-S-OsSD1 vector.

Guide editing system PE-P6 ^ΔOsMLH1 RT-M expression vectors total 3, PE-P6 respectively ^ΔOsMLH1 RT-M-OsEPSPS-2、PE-P6 ^ΔOsMLH1 RT-M-OsGRF4-T2、PE-P6 ^ΔOsMLH1 RT-M-OsSD1 vector.

The nucleotide sequence of the PE-P3-RTM-OsALS-3 recombinant expression vector (denoted as PE-P3-1 vector) is shown in sequence 6. Wherein, the 104 th to 2075 th positions of the sequence 6 are the nucleotide sequence of the ZmUbi1 promoter; the 2292-4322 th bit is the coding sequence of M-MLV RT protein, the coding sequence 1 shows the M-MLV RT protein; the 4422-8522 is the coding sequence (without a stop codon) of the Cas9n (H840A) protein, and codes for the Cas9n (H840A) protein shown in the sequence 2; the 8673-8729 bit is the coding sequence of P2A, and the coding sequence 3 shows the protein; positions 8730-9755 are the coding sequence of Hygromycin Phosphotransferase (HPT), the HPT protein shown in the coding sequence 4; positions 9762-10016 are Nos terminator sequences; the nucleotide sequence of the promoter OsU a at positions 10025-10490, the esgRNA target sequence for generating a non-coding strand incision at positions 10491-10510, the esgRNA framework sequence for generating a non-coding strand incision at positions 10511-10596, and the Poly T at positions 10597-10605; the nucleotide sequence of the promoter OsU is 10606-10986, the target sequence of the pegRNA-01 is 10987-11006, the skeleton sequence of the esgRNA corresponding to the pegRNA-01 is 11007-11092, the RT & PBS sequence on the pegRNA-01 is 11093-11119, and the Poly T is 11120-11127. The esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01, which are corresponding to the pegRNA-01 in the PE-P3-RTM-OsALS-3 recombinant expression vector and generate non-coding strand cuts, are shown in Table 1.

The nucleotide sequence of the PE-P3-RTM-OsEPSPS-2 recombinant expression vector (marked as PE-P3-2 vector) is obtained by replacing the esgRNA target sequence which generates a non-coding chain notch and corresponds to the pegRNA-01 in the PE-P3-1 vector, the esgRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 with the esgRNA target sequence which generates a non-coding chain notch and corresponds to the pegRNA-02, the pegRNA-02 target sequence and the RT & PBS sequence on the pegRNA-02 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P3-RTM-OsGRF4-T2 recombinant expression vector (marked as PE-P3-3 vector) is obtained by replacing the esgRNA target sequence which generates a non-coding chain notch and corresponds to the pegRNA-01 in the PE-P3-1 vector, the esgRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 with the esgRNA target sequence which generates a non-coding chain notch and corresponds to the pegRNA-03, the pegRNA-03 target sequence and the RT & PBS sequence on the pegRNA-03 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P3-RTM-OsSD1 recombinant expression vector (marked as PE-P3-4 vector) is obtained by replacing the esgRNA target sequence which generates a non-coding chain notch and corresponds to the pegRNA-01 in the PE-P3-1 vector, the esgRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 with the esgRNA target sequence which generates a non-coding chain notch and corresponds to the pegRNA-04, the pegRNA-04 target sequence and the RT & PBS sequence on the pegRNA-04 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P3-RTM-OsCold1 recombinant expression vector (marked as PE-P3-5 vector) is obtained by replacing the esgRNA target sequence which generates a non-coding strand cut and corresponds to the pegRNA-01 in the PE-P3-1 vector, the esgRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 with the esgRNA target sequence which generates a non-coding strand cut and corresponds to the pegRNA-05, the pegRNA-05 target sequence and the RT & PBS sequence on the pegRNA-05 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P3-RTM-OsALS-1 recombinant expression vector (marked as PE-P3-6 vector) is obtained by replacing the esgRNA target sequence which generates a non-coding strand incision and corresponds to the pegRNA-01 in the PE-P3-1 vector, the esgRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 with the esgRNA target sequence which generates a non-coding strand incision and corresponds to the pegRNA-06, the pegRNA-06 target sequence and the RT & PBS sequence on the pegRNA-06 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P3-RTM-OsACC-1 recombinant expression vector (marked as PE-P3-7 vector) is obtained by replacing the esgRNA target sequence which generates a non-coding strand incision and corresponds to the pegRNA-01 in the PE-P3-1 vector, the esgRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 with the esgRNA target sequence which generates a non-coding strand incision and corresponds to the pegRNA-07, the pegRNA-07 target sequence and the RT & PBS sequence on the pegRNA-07 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P3-RTM-OsGS3 recombinant expression vector (marked as PE-P3-8 vector) is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 corresponding to the pegRNA-01 in the PE-P3-1 vector with the esgRNA target sequence, the pegRNA-08 target sequence and the RT & PBS sequence on the pegRNA-08 corresponding to the pegRNA-08 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P4-RTM-OsALS-3 recombinant expression vector (denoted as PE-P4-1 vector) is obtained by deleting the nucleotide sequence of the OsU a promoter, the esgRNA target sequence for generating a non-coding strand cut, the esgRNA framework sequence for generating a non-coding strand cut and the Poly T sequence at positions 10597-10605 in the PE-P3-1 vector, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P4-RTM-OsEPSPS-2 recombinant expression vector (marked as PE-P4-2 vector) is obtained by replacing the target sequence of the pegRNA-01 and the RT & PBS sequence on the pegRNA-01 in the PE-P4-1 vector with the target sequence of the pegRNA-02 and the RT & PBS sequence on the pegRNA-02 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P4-RTM-OsGRF4-T2 recombinant expression vector (marked as PE-P4-3 vector) is obtained by replacing the target sequence of the pegRNA-01 and the RT & PBS sequence on the pegRNA-01 in the PE-P4-1 vector with the target sequence of the pegRNA-03 and the RT & PBS sequence on the pegRNA-03 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P4-RTM-OsSD1 recombinant expression vector (marked as PE-P4-4 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P4-1 vector with the pegRNA-04 target sequence and the RT & PBS sequence on the pegRNA-04 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P4-RTM-OsCold1 recombinant expression vector (marked as PE-P4-5 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P4-1 vector with the pegRNA-05 target sequence and the RT & PBS sequence on the pegRNA-05 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P4-RTM-OsALS-1 recombinant expression vector (marked as PE-P4-6 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P4-1 vector with the pegRNA-06 target sequence and the RT & PBS sequence on the pegRNA-06 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P4-RTM-OsACC-1 recombinant expression vector (marked as PE-P4-7 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P4-1 vector with the pegRNA-07 target sequence and the RT & PBS sequence on the pegRNA-07 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P4-RTM-OsGS3 recombinant expression vector (marked as PE-P4-8 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P4-1 vector with the pegRNA-08 target sequence and the RT & PBS sequence on the pegRNA-08 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P5-RTM-OsALS-3 recombinant expression vector (denoted as PE-P5-1 vector) is shown in sequence 7. Wherein, the 104 th to 2075 th positions of the sequence 7 are the nucleotide sequence of the ZmUbi1 promoter; the 2292-4322 th bit is the coding sequence of M-MLV RT protein, the coding sequence 1 shows the M-MLV RT protein; the 4422-8522 positions are the coding sequence (without a stop codon) of the Cas9maxn protein, and the coding sequence 5 shows the Cas9maxn protein; the 8673-8729 bit is the coding sequence of P2A, and the coding sequence 3 shows the protein; the 8730-9755 bit is the coding sequence of HPT, and the HPT protein shown in the coding sequence 4; positions 9762-10016 are Nos terminator sequences; nucleotide sequence of E35s promoter at 10025-10461 position; nucleotide sequence of cmyl cv promoter at position 10462-10920; 10932-11270 are the nucleotide sequences of the OsU promoter; nucleotide sequence of tRNA at 11271-11347; the 11348-11367 is a target sequence of pegRNA-01, the 11368-11453 is an esgRNA skeleton sequence corresponding to the pegRNA-01, the 11454-11478 is an RT & PBS sequence on the pegRNA-01, and the 11479-11546 is a nucleotide sequence of HDV (the HDV is combined with tRNA and can be used for cutting the sgRNA); positions 11547-11554 are Poly T; the HSP terminator sequence is located in positions 11555-11825.

The nucleotide sequence of the PE-P5-RTM-OsEPSPS-2 recombinant expression vector (marked as PE-P5-2 vector) is obtained by replacing the target sequence of the pegRNA-01 and the RT & PBS sequence on the pegRNA-01 in the PE-P5-1 vector with the target sequence of the pegRNA-02 and the RT & PBS sequence on the pegRNA-02 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P5-RTM-OsGRF4-T2 recombinant expression vector (marked as PE-P5-3 vector) is obtained by replacing the target sequence of the pegRNA-01 and the RT & PBS sequence on the pegRNA-01 in the PE-P5-1 vector with the target sequence of the pegRNA-03 and the RT & PBS sequence on the pegRNA-03 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P5-RTM-OsSD1 recombinant expression vector (marked as PE-P5-4 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P5-1 vector with the pegRNA-04 target sequence and the RT & PBS sequence on the pegRNA-04 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P5-RTM-OsCold1 recombinant expression vector (marked as PE-P5-5 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P5-1 vector with the pegRNA-05 target sequence and the RT & PBS sequence on the pegRNA-05 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P5-RTM-OsALS-1 recombinant expression vector (marked as PE-P5-6 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P5-1 vector with the pegRNA-06 target sequence and the RT & PBS sequence on the pegRNA-06 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P5-RTM-OsACC-1 recombinant expression vector (marked as PE-P5-7 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P5-1 vector with the pegRNA-07 target sequence and the RT & PBS sequence on the pegRNA-07 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P5-RTM-OsGS3 recombinant expression vector (marked as PE-P5-8 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P5-1 vector with the pegRNA-08 target sequence and the RT & PBS sequence on the pegRNA-08 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P6-RTM-OsALS-3 recombinant expression vector (denoted as PE-P6-1 vector) is obtained by inserting 8-bp linker and tevopreQ1 motif (sequence 8) of pegRNA-01 target between RT & PBS sequence and HDV sequence of the PE-P5-1 vector, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P6-RTM-OsEPSPS-2 recombinant expression vector (marked as PE-P6-2 vector) is obtained by replacing the target sequence of the pegRNA-01 and the RT & PBS sequence on the pegRNA-01 in the PE-P6-1 vector with the target sequence of the pegRNA-02 and the RT & PBS sequence on the pegRNA-02 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P6-RTM-OsGRF4-T2 recombinant expression vector (marked as PE-P6-3 vector) is obtained by replacing the target sequence of the pegRNA-01 and the RT & PBS sequence on the pegRNA-01 in the PE-P6-1 vector with the target sequence of the pegRNA-03 and the RT & PBS sequence on the pegRNA-03 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P6-RTM-OsSD1 recombinant expression vector (marked as PE-P6-4 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P6-1 vector with the pegRNA-04 target sequence and the RT & PBS sequence on the pegRNA-04 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P6-RTM-OsCold1 recombinant expression vector (marked as PE-P6-5 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P6-1 vector with the pegRNA-05 target sequence and the RT & PBS sequence on the pegRNA-05 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P6-RTM-OsALS-1 recombinant expression vector (marked as PE-P6-6 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P6-1 vector with the pegRNA-06 target sequence and the RT & PBS sequence on the pegRNA-06 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P6-RTM-OsACC-1 recombinant expression vector (marked as PE-P6-7 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P6-1 vector with the pegRNA-07 target sequence and the RT & PBS sequence on the pegRNA-07 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P6-RTM-OsGS3 recombinant expression vector (marked as PE-P6-8 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P6-1 vector with the pegRNA-08 target sequence and the RT & PBS sequence on the pegRNA-08 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P7-RTM-OsALS-3 recombinant expression vector (marked as PE-P7-1 vector) is obtained by inserting 8-bp linker and mpknot motif (sequence 9) of a pegRNA-01 target point between RT & PBS sequence and HDV sequence of the PE-P5-1 vector, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P7-RTM-OsEPSPS-2 recombinant expression vector (marked as PE-P7-2 vector) is obtained by replacing the target sequence of the pegRNA-01 and the RT & PBS sequence on the pegRNA-01 in the PE-P7-1 vector with the target sequence of the pegRNA-02 and the RT & PBS sequence on the pegRNA-02 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P7-RTM-OsGRF4-T2 recombinant expression vector (marked as PE-P7-3 vector) is obtained by replacing the target sequence of the pegRNA-01 and the RT & PBS sequence on the pegRNA-01 in the PE-P7-1 vector with the target sequence of the pegRNA-03 and the RT & PBS sequence on the pegRNA-03 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P7-RTM-OsSD1 recombinant expression vector (marked as PE-P7-4 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P7-1 vector with the pegRNA-04 target sequence and the RT & PBS sequence on the pegRNA-04 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P7-RTM-OsCold1 recombinant expression vector (marked as PE-P7-5 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P7-1 vector with the pegRNA-05 target sequence and the RT & PBS sequence on the pegRNA-05 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P7-RTM-OsALS-1 recombinant expression vector (marked as PE-P7-6 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P7-1 vector with the pegRNA-06 target sequence and the RT & PBS sequence on the pegRNA-06 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P7-RTM-OsACC-1 recombinant expression vector (marked as PE-P7-7 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P7-1 vector with the pegRNA-07 target sequence and the RT & PBS sequence on the pegRNA-07 respectively, and keeping other sequences unchanged.

The nucleotide sequence of the PE-P7-RTM-OsGS3 recombinant expression vector (marked as PE-P7-8 vector) is obtained by replacing the pegRNA-01 target sequence and the RT & PBS sequence on the pegRNA-01 in the PE-P7-1 vector with the pegRNA-08 target sequence and the RT & PBS sequence on the pegRNA-08 respectively, and keeping other sequences unchanged.

PE-P6 ^OE ^hMLHdn RT-S-OsEPSPS-2 recombinant expression vector (named PE-P6) ^OE ^hMLHdn RT-S-1 vector) is an expression cassette comprising an OsAct1-01 promoter sequence (SEQ ID NO: 10), an hMLH1dn gene sequence (SEQ ID NO: 11) and a Nos terminator sequence in this order inserted between the Nos terminator sequence and E35S promoter sequence of PE-P5-1 vector, and a pegRNA-01 target sequence and RT on pegRNA-01 &PBS sequence is replaced by pegRNA-13 target sequence and RT on pegRNA-13 respectively&PBS sequence, and at RT&8-bp linker and tevopreQ1 motif (SEQ ID NO: 9) of pegRNA-13 target were inserted between PBS sequence and HDV sequence, and maintainedOther sequences were obtained after the remaining sequences were unchanged.

PE-P6 ^OE ^hMLHdn RT-S-OsGRF4-T2 recombinant expression vector (denoted as PE-P6) ^OE ^hMLHdn RT-S-2) is to use PE-P6 ^OE ^hMLHdn PegRNA-13 target sequence in RT-S-1 vector and RT on pegRNA-13&PBS sequence is replaced by the target sequence of pegRNA-14 and RT on pegRNA-14 respectively&PBS sequence, and the other sequences were kept unchanged.

PE-P6 ^OE ^hMLHdn RT-S-OsSD1 recombinant expression vector (denoted as PE-P6) ^OE ^hMLHdn RT-S-3) is to use PE-P6 ^OE ^hMLHdn PegRNA-12 target sequence in RT-S-1 vector and RT on pegRNA-12&PBS sequence is replaced by pegRNA-15 target sequence and RT on pegRNA-15 respectively&PBS sequence, and the other sequences were kept unchanged.

PE-P6 ^OE ^hMLHdn RT-M-OsEPSPS-2 recombinant expression vector (named PE-P6- ^OE ^hMLHdn RT-M-1 vector) is an expression cassette comprising an OsAct1-01 promoter sequence (SEQ ID NO: 10), an hMLH1dn gene sequence (SEQ ID NO: 11) and a Nos terminator sequence in this order inserted between the Nos terminator sequence and E35s promoter sequence of PE-P5-1 vector, and a pegRNA-01 target sequence and RT on pegRNA-01 &PBS sequence is replaced by pegRNA-02 target sequence and RT on pegRNA-02&PBS sequence, and at RT&8-bp linker and tevopreQ1 motif (sequence 9) of pegRNA-13 target point are inserted between PBS sequence and HDV sequence, and other sequences are kept unchanged to obtain the sequence.

PE-P6 ^OE ^hMLHdn RT-M-OsGRF4-T2 recombinant expression vector (denoted as PE-P6) ^OE ^hMLHdn RT-M-2 vector) is to use PE-P6 ^OE ^hMLHdn PegRNA-02 target sequence in RT-M-1 vector and RT on pegRNA-02&PBS sequence is replaced by pegRNA-03 target sequence and RT on pegRNA-03&PBS sequence, and the other sequences were kept unchanged.

PE-P6 ^OE ^hMLHdn RT-M-OsSD1 recombinant expression vector (denoted as PE-P6) ^OE ^hMLHdn RT-M-3 vector) is to use PE-P6 ^OE ^hMLHdn PegRNA-02 target sequence in RT-M-1 vector and RT on pegRNA-02&PBS sequence is replaced by the pegRNA-04 target sequence and RT on pegRNA-04&PBS sequence, and the other sequences were kept unchanged.

PE-P6 ^ΔOsMLH1 RT-S-OsEPSPS-2 recombinant expression vector (named PE-P6) ^ΔOsMLH1 RT-S-1 vector) is prepared by adding PE-P6 to the nucleotide sequence ^OE ^hMLHdn The expression cassette consisting of the OsAct1-01 promoter sequence (sequence 10), the hMLH1dn gene sequence (sequence 11) and the Nos terminator sequence in the RT-S-1 vector is replaced by a DNA fragment consisting of a OsU a promoter and a DNA molecule shown by the sequence 12 (tRNA+MLH 1-T1 target sequence+esgRNA backbone sequence+tRNA+MLH 1-T2 target sequence+esgRNA backbone sequence+PolyT) in sequence, and other sequences are kept unchanged to obtain the sequence. In sequence 12, tRNA is at positions 1-77, MLH1-T1 is at positions 78-97, esgRNA backbone sequence is at positions 98-183, tRNA is at positions 184-260, MLH1-T2 is at positions 261-280, esgRNA backbone sequence is at positions 281-366, and PolyT is at positions 367-372.

PE-P6 ^ΔOsMLH1 RT-S-OsGRF4-T2 recombinant expression vector (denoted as PE-P6) ^ΔOsMLH1 RT-S-2 vector) is a vector which is prepared by combining PE-P6 ^ΔOsMLH1 PegRNA-13 target sequence in RT-S-1 vector and RT on pegRNA-13&PBS sequence is replaced by the target sequence of pegRNA-14 and RT on pegRNA-14 respectively&PBS sequence, and the other sequences were kept unchanged.

PE-P6 ^ΔOsMLH1 RT-S-OsSD1 recombinant expression vector (denoted as PE-P6) ^ΔOsMLH1 RT-S-3 vector) is a vector which is prepared from PE-P6 ^ΔOsMLH1 PegRNA-13 target sequence in RT-S-1 vector and RT on pegRNA-13&PBS sequence is replaced by pegRNA-15 target sequence and RT on pegRNA-15 respectively&PBS sequence, and the other sequences were kept unchanged.

PE-P6 ^ΔOsMLH1 RT-M-OsEPSPS-2 recombinant expression vector (named PE-P6) ^ΔOsMLH1 RT-M-1 vector) is prepared by adding PE-P6 to the nucleotide sequence ^ΔOsMLH1 PegRNA-13 target sequence in RT-S-1 vector and RT on pegRNA-13&PBS sequence is replaced by pegRNA-02 target sequence and RT on pegRNA-02&PBS sequence, and other sequences are maintainedThe sequence obtained after the column was unchanged.

PE-P6 ^ΔOsMLH1 RT-M-OsGRF4-T2 recombinant expression vector (denoted as PE-P6) ^ΔOsMLH1 RT-M-2 vector) is to use PE-P6 ^ΔOsMLH1 PegRNA-02 target sequence in RT-M-1 vector and RT on pegRNA-02&PBS sequence is replaced by pegRNA-03 target sequence and RT on pegRNA-03&PBS sequence, and the other sequences were kept unchanged.

PE-P6 ^ΔOsMLH1 RT-M-OsSD1 recombinant expression vector (denoted as PE-P6) ^ΔOsMLH1 RT-M-3 vector) is to use PE-P6 ^ΔOsMLH1 PegRNA-02 target sequence in RT-M-1 vector and RT on pegRNA-02&PBS sequence is replaced by the pegRNA-04 target sequence and RT on pegRNA-04&PBS sequence, and the other sequences were kept unchanged.

The esgRNA target sequence and RT & PBS sequence on the pegRNA in each vector, and the 8-bp linker sequence are shown in Table 1.

TABLE 1

2. Rice resistant callus and acquisition of positive T0 seedlings

8 expression vectors in each of PE-P3, PE-P4, PE-P5, PE-P6 and PE-P7 of the guidance editing system for construction in the step one, and the guidance editing system PE-P6 ^ΔOsMLH1 RT-S and PE-P6 ^ΔOsMLH1 3 expression vectors in RT-M, 46 recombinant expression vectors in total, were operated according to the following steps 1-9, respectively:

1. the vector was introduced into Agrobacterium EHA105 (product of Shanghai Di Biotechnology Co., ltd.; CAT#: AC 1010) to obtain recombinant Agrobacterium.

2. Recombinant Agrobacterium was cultured using medium (YEP medium containing 50. Mu.g/mL kanamycin and 25. Mu.g/mL rifampicin), shake cultured at 28℃and 150rpm to OD ₆₀₀ 1.0-2.0, centrifuging at 10000rpm for 1min at room temperature, and collecting the solution (glucose and sucrose are replaced by sugar in N6 liquid culture medium, and the concentrations of glucose and sucrose in the solution are 10 g- L and 20 g/L) re-suspension of the cells and dilution to OD ₆₀₀ And (3) obtaining the agrobacterium infection solution with the concentration of 0.2.

3. Removing shells of mature seeds of a rice variety Japanese sunny day, putting the mature seeds into a 100mL triangular flask, adding 70% (v/v) ethanol aqueous solution for soaking for 30sec, putting the mature seeds into 25% (v/v) sodium hypochlorite aqueous solution, vibrating and sterilizing for 30min at 120rpm, washing with sterile water for 3 times, sucking water by using filter paper, putting seed embryos downwards on an N6 solid medium, and culturing in dark at 28 ℃ for 4-6 weeks to obtain rice calli.

4. After the step 3 is completed, the rice callus is soaked in agrobacterium infection solution A (the agrobacterium infection solution A is a liquid obtained by adding acetosyringone into the agrobacterium infection solution, the addition amount of the acetosyringone satisfies the volume ratio of the acetosyringone to the agrobacterium infection solution is 25 mu L:50 mL) for 10min, and then the rice callus is placed on a culture dish (containing about 200mL of infection solution without agrobacterium) paved with two layers of sterilization filter paper, and is subjected to dark culture at 21 ℃ for 1 day.

5. And (3) putting the rice callus obtained in the step (4) on a recovery culture medium, and carrying out dark culture at 25-28 ℃ for 3 days.

6. And (3) taking the rice callus obtained in the step (5), placing the rice callus on a screening culture medium, and culturing the rice callus in dark at 28 ℃ for 2 weeks.

7. And (3) taking the rice callus obtained in the step (6), and placing the rice callus on a screening culture medium again, and carrying out dark culture at 28 ℃ for 2 weeks to obtain the rice resistant callus.

8. And (3) placing the rice resistant callus obtained in the step (7) on a differentiation medium, culturing for about 1 month at 25 ℃ by illumination, transferring the differentiated plantlet onto a rooting medium, and culturing for 2 weeks at 25 ℃ by illumination to obtain the rice T0 plantlet.

9. Extracting genome DNA of the obtained rice T0 seedling by taking the obtained genome DNA as a template and carrying out PCR amplification by adopting a primer pair consisting of a primer F (5'-TACTCTCATCCACCAGTCCATC-3') and a primer R (5'-GATGTTGGCGACCTCGTAT-3') to obtain a PCR amplification product, wherein the PE-P3-RTM-OsALS-3, PE-P3-RTM-OsALS-2, PE-P3-RTM-OsEPSPS-2, PE-P3-RTM-OsGRF4-T2, PE-P3-RTM-OsSD1, PE-P3-RTM-Oscold1, PE-P3-RTM-OsACC-1 and PE-P3-RTM-OsGS34 are recombined; the PCR amplified product was subjected to agarose gel electrophoresis, and then judged as follows: if the PCR amplification product contains a DNA fragment of about 973bp, the corresponding rice T0 seedling is a rice positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973bp, the corresponding rice T0 seedling is not a rice positive T0 seedling.

Extracting genome DNA of the obtained rice T0 seedling and taking the genome DNA as a template of the recombinant expression vectors of PE-P4-RTM-OsALS-3, PE-P4-RTM-OsEPSPS-2, PE-P4-RTM-OsGRF4-T2, PE-P4-RTM-OsSD1, PE-P4-RTM-Oscold1, PE-P4-RTM-OsALS-1 and PE-P4-RTM-OsGS3, and carrying out PCR amplification by adopting a primer pair consisting of a primer F (5'-TACTCTCATCCACCAGTCCATC-3') and a primer R (5'-GATGTTGGCGACCTCGTAT-3') to obtain a PCR amplification product; the PCR amplified product was subjected to agarose gel electrophoresis, and then judged as follows: if the PCR amplification product contains a DNA fragment of about 973bp, the corresponding rice T0 seedling is a rice positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973bp, the corresponding rice T0 seedling is not a rice positive T0 seedling.

Extracting genome DNA of the obtained rice T0 seedling by taking the obtained genome DNA as a template and carrying out PCR amplification by adopting a primer pair consisting of a primer F (5'-TACTCTCATCCACCAGTCCATC-3') and a primer R (5'-GATGTTGGCGACCTCGTAT-3') to obtain a PCR amplification product, wherein the PE-P5-RTM-OsALS-3, PE-P5-RTM-OsEPSPS-2, PE-P5-RTM-OsGRF4-T2, PE-P5-RTM-OsSD1, PE-P5-RTM-Oscold1, PE-P5-RTM-OsACC-1 and PE-P5-RTM-OsGS3 are recombined; the PCR amplified product was subjected to agarose gel electrophoresis, and then judged as follows: if the PCR amplification product contains a DNA fragment of about 973bp, the corresponding rice T0 seedling is a rice positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973bp, the corresponding rice T0 seedling is not a rice positive T0 seedling.

Extracting genome DNA of the obtained rice T0 seedling and taking the genome DNA as a template of the recombinant expression vectors of PE-P6-RTM-OsALS-3, PE-P6-RTM-OsEPSPS-2, PE-P6-RTM-OsGRF4-T2, PE-P6-RTM-OsSD1, PE-P6-RTM-Oscold1, PE-P6-RTM-OsALS-1 and PE-P6-RTM-OsGS3, and carrying out PCR amplification by adopting a primer pair consisting of a primer F (5'-TACTCTCATCCACCAGTCCATC-3') and a primer R (5'-GATGTTGGCGACCTCGTAT-3') to obtain a PCR amplification product; the PCR amplified product was subjected to agarose gel electrophoresis, and then judged as follows: if the PCR amplification product contains a DNA fragment of about 973bp, the corresponding rice T0 seedling is a rice positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973bp, the corresponding rice T0 seedling is not a rice positive T0 seedling.

Extracting genome DNA of the obtained rice T0 seedling by taking the obtained genome DNA as a template and carrying out PCR amplification by adopting a primer pair consisting of a primer F (5'-TACTCTCATCCACCAGTCCATC-3') and a primer R (5'-GATGTTGGCGACCTCGTAT-3') to obtain a PCR amplification product, wherein the PE-P7-RTM-OsALS-3, PE-P7-RTM-OsEPSPS-2, PE-P7-RTM-OsGRF4-T2, PE-P7-RTM-OsSD1, PE-P7-RTM-Oscold1, PE-P7-RTM-OsALS-1 and PE-P7-RTM-OsGS3 are recombined; the PCR amplified product was subjected to agarose gel electrophoresis, and then judged as follows: if the PCR amplification product contains a DNA fragment of about 973bp, the corresponding rice T0 seedling is a rice positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973bp, the corresponding rice T0 seedling is not a rice positive T0 seedling.

For PE-P6 ^OE ^hMLHdn RT-S-OsEPSPS-2、PE-P6 ^OE ^hMLHdn RT-S-OsGRF4-T2、PE-P6 ^OE ^hMLHdn RT-S-OsSD1 recombinant expression vector, extracting genome DNA of obtained rice T0 seedling and using the genome DNA as template, and adopting primer F (5' -TACTCTCATC)CACCAGTCCATC-3 ') and primer R (5'-GATGTTGGCGACCTCGTAT-3') to obtain PCR amplified products; the PCR amplified product was subjected to agarose gel electrophoresis, and then judged as follows: if the PCR amplification product contains a DNA fragment of about 973bp, the corresponding rice T0 seedling is a rice positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973bp, the corresponding rice T0 seedling is not a rice positive T0 seedling.

For PE-P6 ^OE ^hMLHdn RT-M-OsEPSPS-2、PE-P6 ^OE ^hMLHdn RT-M-OsGRF4-T2、PE-P6 ^OE ^hMLHdn Extracting genome DNA of the obtained rice T0 seedling and taking the genome DNA as a template, and carrying out PCR amplification by using a primer pair consisting of a primer F (5'-TACTCTCATCCACCAGTCCATC-3') and a primer R (5'-GATGTTGGCGACCTCGTAT-3') to obtain a PCR amplification product; the PCR amplified product was subjected to agarose gel electrophoresis, and then judged as follows: if the PCR amplification product contains a DNA fragment of about 973bp, the corresponding rice T0 seedling is a rice positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973bp, the corresponding rice T0 seedling is not a rice positive T0 seedling.

For PE-P6 ^ΔOsMLH1 RT-S-OsEPSPS-2、PE-P6 ^ΔOsMLH1 RT-S-OsGRF4-T2、PE-P6 ^ΔOsMLH1 Extracting genome DNA of the obtained rice T0 seedling and taking the genome DNA as a template, and carrying out PCR amplification by using a primer pair consisting of a primer F (5'-TACTCTCATCCACCAGTCCATC-3') and a primer R (5'-GATGTTGGCGACCTCGTAT-3') to obtain a PCR amplification product; the PCR amplified product was subjected to agarose gel electrophoresis, and then judged as follows: if the PCR amplification product contains a DNA fragment of about 973bp, the corresponding rice T0 seedling is a rice positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973bp, the corresponding rice T0 seedling is not a rice positive T0 seedling.

For PE-P6 ^ΔOsMLH1 RT-M-OsEPSPS-2、PE-P6 ^ΔOsMLH1 RT-M-OsGRF4-T2、PE-P6 ^ΔOsMLH1 The RT-M-OsSD1 recombinant expression vector is used for extracting the obtained genome DNA of the rice T0 seedling and taking the genome DNA as a template, and the method adoptsPerforming PCR amplification on a primer pair consisting of a primer F (5'-TACTCTCATCCACCAGTCCATC-3') and a primer R (5'-GATGTTGGCGACCTCGTAT-3') to obtain a PCR amplification product; the PCR amplified product was subjected to agarose gel electrophoresis, and then judged as follows: if the PCR amplification product contains a DNA fragment of about 973bp, the corresponding rice T0 seedling is a rice positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973bp, the corresponding rice T0 seedling is not a rice positive T0 seedling.

3. Analysis of results of editing efficiency in Rice callus and T0 seedlings

1. And (3) in PE-P3-PE-P7, randomly selecting 32 resistant calli obtained in the step (7) in the step (II) for each vector, extracting DNA, randomly mixing 8 calli DNA, and finally obtaining 4 mixed DNA, namely dividing into 4 groups. Taking the mixed DNA as a template, and carrying out PCR amplification on sequences near the OsALS-3 target point by adopting a primer F (5'-CTCCAGGGCCATACTTGTTG-3') and a primer R (5'-TGGGTCATTCAGGTCAAACA-3') to obtain a first-round PCR amplification product; for an OsEPSPS-2 target, performing PCR amplification on a sequence near the OsEPSPS-2 target by adopting a primer F (5'-GGCTCTCTGTGGAAGCAGAT-3') and a primer R (5'-CATACGTTGCATTTCCACCA-3') to obtain a first round PCR amplification product; for the OsGRF4-T2 target, carrying out PCR amplification on sequences near the OsGRF4-T2 target by adopting a primer F (5'-CCATTTTCTTGGCTCCAGTG-3') and a primer R (5'-CTGCTCCAGCTCCTCGTACT-3') to obtain a first round of PCR amplification product; for an OsSD1 target, carrying out PCR amplification on sequences near the OsSD1 target by adopting a primer F (5'-CGTGTCCGGCTACACCAG-3') and a primer R (5'-AATGTCGTCCACCATCGTTT-3') to obtain a first-round PCR amplification product; for the OsCold1 target, carrying out PCR amplification on sequences near the Oscold1 target by adopting a primer F (5'-CTATGCTATGCGTGCCAATC-3') and a primer R (5'-CCTCTCCATCTCCATTTTGG-3') to obtain a first-round PCR amplification product; for an OsALS-1 target, carrying out PCR amplification on a sequence near the OsALS-1 target by adopting a primer F (5'-TTGAGAACCTCCCTGTGAAG-3') and a primer R (5'-TGAACCCCTTAGCAATAGTCACA-3') to obtain a first round PCR amplification product; for an OsACC-1 target, performing PCR amplification by using a primer F (5'-TCTACATTCCCATGGCTGC-3') and a primer R (5'-CCAGAACATTGCCTTTTGC-3') to obtain a first round PCR amplification product; for the OsGS3 target, a primer F (5'-AATCATGGATTTTGGCTTGG-3') and a primer R (5'-GCTTCTCCGATGAACTGCTT-3') are adopted to carry out PCR amplification on sequences near the OsGS3 target, so that a first round of PCR amplification product is obtained. The PCR products of the first round are used as templates, different forward and reverse bar codes are added into the ends of the PCR products to construct a library, a mixed library is formed, sequencing was performed using a MiSeq high throughput sequencing platform, with each mixed library sequenced to a depth of more than 5000X (Optimus in the field of Prime Biotechnology Co.). Sequencing results were analyzed for each pegRNA region by CRISPResso2 (https:// doi. Org/10.1038/s 41587-019-0032-3) and the editing efficiency of the guided editing system in rice calli was calculated. The efficiency of editing in the rice calli by the guidance editing system was an average of 4 groups of the ratio of the number of reads detected with all mutation sites to the total number of reads.

The results are shown in FIG. 3. The results show that in PE-P3-PE-P7, the editing efficiency of the OsALS-3 target spot in the rice callus is respectively as follows: 23.7%, 15.85%, 10.375%, 17.775% and 0; editing efficiency of the OsEPSPS-2 target points in the rice calluses is respectively as follows: 6.75%, 7.4%, 12.675%, 32.525%, and 0.925%; editing efficiency of the OsGRF4-T2 target spot in the rice callus is respectively as follows: 0.5%, 0, 2.9%, 5.425% and 0; editing efficiency of the OsSD1 target in the rice callus is respectively as follows: 4.275%, 1%, 27.7%, 27.175% and 0; editing efficiency of the OsCold1 target in the rice callus is respectively as follows: 0. 0, 19.2% and 0; editing efficiency of the OsALS-1 target spot in the rice callus is respectively as follows: 26.375%, 5.1%, 0.35%, 27.425% and 0.575%; editing efficiency of the OsACC-1 target spot in the rice callus is respectively as follows: 0.45%, 0.275%, 3.95%, 36.975% and 1.925%; editing efficiency of the OsGS3 target spot in the rice callus is respectively as follows: 2.6%, 2.5%, 0, 32.25% and 1.625%. In conclusion, the editing efficiency of the rice target in callus is greatly improved by guiding the editing system PE-P6.

2. In PE-P3-PE-P7, taking the genome DNA of the rice positive T0 seedlings obtained in the step 9 in the step one as a template for each vector, and carrying out PCR amplification on the OsALS-3 target spot by adopting a primer pair to obtain a first round of PCR amplification product; for an OsEPSPS-2 target, carrying out PCR amplification on the OsEPSPS-2 by adopting a primer pair to obtain a first round of PCR amplification product; for the OsGRF4-T2 target, carrying out PCR amplification on the OsGRF4-T2 by adopting a primer pair to obtain a first round PCR amplification product; for an OsSD1 target, carrying out PCR amplification on the OsSD1 by adopting a primer to obtain a first round PCR amplification product; for the OsCold1 target, carrying out PCR amplification on the Oscold1 by adopting a primer to obtain a first round of PCR amplification product; for an OsALS-1 target, carrying out PCR amplification on the OsALS-1 by adopting a primer to obtain a first round PCR amplification product; for an OsACC-1 target, carrying out PCR amplification on the OsACC-1 by adopting a primer to obtain a first round of PCR amplification product; and for the OsGS3 target, carrying out PCR amplification on the OsGS3 by adopting a primer to obtain a first round of PCR amplification product. The first round of PCR products are used as templates, different forward and reverse barcodes are added to the ends of the PCR products for library construction to form mixed libraries, and the mixed libraries are sequenced by using a Miseq high-throughput sequencing platform, wherein the sequencing depth of each mixed library is more than 1000X (the engineering Co., ltd.). Analysis was performed for each pegRNA region and the editing efficiency of the guided editing system in rice T0 seedlings was calculated. Editing efficiency of the pilot editing system in rice T0 seedlings = number of positive T0 seedlings mutated at all mutation sites/total number of positive T0 seedlings analyzed x 100%.

The results are shown in Table 2. The results show that in PE-P3-PE-P7, the editing efficiency of the OsALS-3 target spot in the rice T0 seedling is respectively as follows: 68.0%, 60%, 39.3%, 73% and 0; editing efficiency of OsEPSPS-2 targets in the rice T0 seedlings is respectively as follows: 79.7%, 54.0%, 70.4%, 91.5%, and 0; editing efficiency of OsGRF4-T2 targets in the rice T0 seedlings is respectively as follows: 20.6%, 1.7%, 19.6%, 54.5% and 11.3%; editing efficiency of OsSD1 targets in the rice T0 seedlings is respectively as follows: 2.2%, 2.9%, 37.3%, 48.6% and 3.3%; editing efficiency of OsCold1 targets in the rice T0 seedlings is respectively as follows: 0. 0, 1.4%, 60.2% and 2.7%; editing efficiency of OsALS-1 targets in the rice T0 seedlings is respectively as follows: 76.0%, 33.7%, 2.4%, 50.6% and 0; editing efficiency of the OsACC-1 target spot in the rice T0 seedling is respectively as follows: 4.2%, 14.9%, 18.8%, 58.3% and 37.5%; editing efficiency of the OsGS3 target spot in the rice T0 seedling is respectively as follows: 3.5%, 18.6%, 5.3%, 56.3% and 3.3%. In summary, compared with PE-P3, PE-P4, PE-P5 and PE-P7, in the rice T0 seedlings, the efficiency of 8 rice targets after being edited by the guiding editing system PE-P6 is highest.

3. In PE-P6 ^OE ^hMLHdn RT-S、PE-P6 ^OE ^hMLHdn RT-M、PE-P6 ^ΔOsMLH1 RT-S and PE-P6 ^ΔOsMLH1 In RT-M, each vector takes the genome DNA of the positive T0 rice seedling obtained in the step 9 in the step one as a template. For an OsEPSPS-2 target, performing PCR amplification on an OsEPSPS-2 target accessory sequence by adopting a primer F (5'-GGCTCTCTGTGGAAGCAGAT-3') and a primer R (5'-CATACGTTGCATTTCCACCA-3') to obtain a first round PCR amplification product; for the OsGRF4-T2 target, carrying out PCR amplification on sequences near the OsGRF4-T2 target by adopting a primer F (5'-CCATTTTCTTGGCTCCAGTG-3') and a primer R (5'-CTGCTCCAGCTCCTCGTACT-3') to obtain a first round of PCR amplification product; for the OsSD1 target, a primer F (5'-CGTGTCCGGCTACACCAG-3') and a primer R (5'-AATGTCGTCCACCATCGTTT-3') are adopted to carry out PCR amplification on sequences near the OsSD1 target, so that a first round PCR amplification product is obtained. The first round of PCR products are used as templates, different forward and reverse barcodes are added to the ends of the PCR products for library construction to form mixed libraries, and the mixed libraries are sequenced by using a Miseq high-throughput sequencing platform, wherein the sequencing depth of each mixed library is more than 1000X (the engineering Co., ltd.). Analysis was performed for each pegRNA region and the editing efficiency of the guided editing system in rice T0 seedlings was calculated. Editing efficiency of the pilot editing system in rice T0 seedlings = number of positive T0 seedlings mutated at all mutation sites/total number of positive T0 seedlings analyzed x 100%.

The results are shown in Table 3. The results show that in PE-P6 and PE-P6 ^OE ^hMLHdn RT-S、PE-P6 ^OE ^hMLHdn RT-M、PE-P6 ^ΔOsMLH1 RT-S and PE-P6 ^ΔOsMLH1 In RT-M, the editing efficiency of OsEPSPS-2 targets in rice T0 seedlings is respectively as follows: 84.3%, 60.5.8%, 77.1%, 93.5% and 92.5%; editing efficiency of OsGRF4-T2 targets in the rice T0 seedlings is respectively as follows: 56.8%, 40.0%, 64.6%, 66.6% and 85.5%; editing efficiency of OsSD1 targets in the rice T0 seedlings is respectively as follows: 44.8%, 64.0%, 73.8%, 64.0% and 86.5%. In conclusion, compared with the guidance editing system PE-P6, the hMLHdn gene is overexpressed and guidedEditing efficiency is not improved; after knocking out the OsMLH1 gene, guiding and editing the system PE-P6 ^ΔOsMLH1 RT-S and PE-P6 ^ΔOsMLH1 The RT-M can further improve the editing efficiency of the target point; and boot editing system PE-P6 ^ΔOsMLH1 The RT-M can improve the editing efficiency of the rice target point to the greatest extent.

TABLE 2

TABLE 3 Table 3

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that it will be apparent to those skilled in the art that several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the scope of the invention.

Claims

1. A kit comprising a fusion protein, esgRNA, and pegRNA;

the esgRNA targets an MLH1 gene target sequence;

the pegRNA sequentially comprises esgRNA', a reverse transcription template sequence, a primer binding site sequence, a connecting sequence and a tevopreQ1 motif; the esgRNA' targets the target sequence of the target gene.

2. The kit of claim 1, wherein:

the esgRNA consists of an MLH1 gene target sequence and an esgRNA skeleton;

or, the esgRNA' consists of a target sequence of a target gene and an esgRNA framework;

alternatively, the pegRNA is expressed driven by a composite promoter; the composite promoter sequentially comprises an E35S promoter, a CmYLCV promoter and a OsU promoter;

or, the nucleotide sequence of the E35S promoter is shown in 10025-10461 positions of the sequence 7;

or, the nucleotide sequence of the CmYLCV promoter is shown in the 10462-10920 positions of the sequence 7;

alternatively, the nucleotide sequence of the OsU3 promoter is shown in positions 10932-11270 of the sequence 7.

3. The kit of claim 1 or 2, wherein: the Cas9 nickase is Cas9maxn;

or, the Cas9maxn is A1) or A2):

A1 Amino acid sequence is a protein shown in sequence 5;

a2 A protein having the same function and obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2.

4. A kit according to any one of claims 1-3, wherein: the reverse transcriptase is M-MLV RT;

or, the M-MLV RT is B1) or B2):

b1 Amino acid sequence is a protein shown in sequence 1;

5. The kit of any one of claims 1-4, wherein: the self-cleaving oligopeptide is a 2A self-cleaving oligopeptide from a viral genome;

or, the 2A self-cleaving oligopeptide from the viral genome is a 2A self-cleaving oligopeptide from porcine teschovirus-1;

or, the amino acid sequence of the 2A self-cleaving oligopeptide from porcine teschovirus-1 is C1) or C2):

c1 Amino acid sequence is a protein shown in sequence 3;

6. The kit of any one of claims 1-5, wherein: the screening agent resistant protein is hygromycin phosphotransferase;

or, the hygromycin phosphotransferase is D1) or D2):

d1 Amino acid sequence is a protein shown in sequence 4;

7. Use of the kit of any one of claims 1-6 in any one of the following S1) -S4):

s1) editing genome sequences of organisms or biological cells;

T1) -T3) method as set forth in any one of:

t1) a method for editing a genomic sequence, comprising the steps of: allowing an organism or biological cell to express the fusion protein of claim 1, the esgRNA of claim 1, and the pegRNA of claim 1;

t2) a method for increasing the efficiency of editing genomic sequences of an organism or a biological cell, comprising the steps of: allowing an organism or biological cell to express the fusion protein of claim 1, the esgRNA of claim 1, and the pegRNA of claim 1;

9. The kit according to any one of claims 1-6 or the use according to claim 7 or the method according to claim 8, characterized in that: the editing of the genomic sequence is a base substitution of the genomic sequence.

10. The kit according to any one of claims 1-6 or the use according to claim 7 or the method according to claim 8, characterized in that: the organism is X1) or X2) or X3) or X4):