A method to improve the efficiency of base editing in a guide editing system

Abstract

The present 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 present invention knocks out the rice MMR repair gene based on the PE-P6 guide editing system to obtain the guide editing system PE-P6 ΔOsMLH1 RT‑S and Guided Editing System PE‑P6 ΔOsMLH1 Compared with the PE-P6 guide editing system, after knocking out the OsMLH1 gene, the guide editing system PE-P6 ΔOsMLH1 RT‑S and PE‑P6 ΔOsMLH1 RT‑M can further improve the editing efficiency of the target; and guide the editing system PE‑P6 ΔOsMLH1 RT‑M maximizes editing efficiency of rice targets.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for improving the base editing efficiency of a guide editing system. Background Art

[0002] In 2019, David Liu's laboratory developed a new versatile genome editing technology, Prime editing (PE), which can accurately achieve 12 types of base substitutions, small insertions and deletions without the need for double-stranded DNA breaks or donor DNA templates. The minimum composition of the PE system is a Cas9 nickase (Cas9n) and Moloney murine leukemia virus reverse transcriptase (M-MLV reverse transcriptase, M-MLV RT), as well as a prime editing guide RNA (pegRNA). The pegRNA contains a spacer region that specifies the target site, a single-guide RNA (sgRNA) scaffold, and a 3' extension. The 3' extension contains a primer binding site (PBS) that is complementary to a portion of the DNA spacer region, and an RT template that encodes the desired editing and downstream genomic sequences. In 2021, an engineered pegRNA (epegRNA) integrated a structured RNA motif into the 3' end of the pegRNA, increasing the efficiency of guided editing in human cells by 3 to 4 times.

[0003] The PE editing efficiency of most target sites in plants is much lower than the 20% to 70% editing efficiency in human cells. In recent years, a number of efforts, mainly including the optimization of PE components, have been used to further improve the application of guide editing in crops. For example, the use of polycistronic tRNA and ribozymes to promote the expression of pegRNA in maize, the introduction of RT templates in rice in coordination with N-terminal reverse transcriptase-cas9n fusion protein for polynucleotide replacement, and the removal of the ribonuclease H domain of M-MLVRT and the addition of viral nucleocapsid proteins with nucleic acid chaperone activity in rice and wheat can increase the flexibility and applicability of PE. However, after the above improvements, the average efficiency of PE in stable transgenic plants is still not high. How to improve the efficiency of PE, especially at the level of stable transformation of crops, remains challenging. Summary of the invention

[0004] An object of the present invention is to provide a complete system.

[0005] The complete system includes a fusion protein, an esgRNA and a pegRNA;

[0006] The fusion protein includes reverse transcriptase, Cas9 nicking enzyme, self-cleaving oligopeptide and screening marker protein in sequence;

[0007] The esgRNA targets the MLH1 gene target sequence;

[0008] The pegRNA includes esgRNA', a reverse transcription template sequence (RT sequence), a primer binding site sequence (PBS sequence), a connection sequence and a tevopreQ1 motif in sequence; the esgRNA' targets the target gene target sequence.

[0009] In the above-mentioned complete system, the esgRNA is composed of the MLH1 gene target sequence and the esgRNA skeleton in sequence.

[0010] Further, the esgRNA is an esgRNA with tRNA, and the tRNA is an RNA molecule obtained by replacing T in positions 1-77 of sequence 12 with U. The esgRNA backbone is an RNA molecule obtained by replacing T in positions 11368-11453 of sequence 7 with U.

[0011] The number of the esgRNAs may be 1 or 2 or more.

[0012] The MLH1 gene sequence is shown in Sequence 13.

[0013] Furthermore, there are two esgRNAs, respectively denoted as esgRNA1 and esgRNA2. The target sequence in esgRNA1 is shown in SEQ ID NO: 78-97, and the target sequence in esgRNA2 is shown in SEQ ID NO: 261-280.

[0014] In the above-mentioned complete system, the pegRNA is composed of the target gene target sequence, the esgRNA backbone, the RT sequence and the PBS sequence, the connecting sequence and the tevopreQ1 motif in sequence.

[0015] The esgRNA backbone is an RNA molecule obtained by replacing T in positions 11368-11453 of sequence 7 with U.

[0016] The RT sequence is the reverse complementary sequence of the 3 'end 3 bases of the target sequence and a continuous genomic sequence thereafter, and the target mutation is introduced therein, and serves as a reverse transcription template for reverse transcriptase to reverse transcribe cDNA, and then serves as a repair template to repair the genomic DNA. The RT sequence size can further be 8-34 bp.

[0017] The PBS sequence (primer binding site sequence) is the reverse complementary sequence of the target sequence from the nth base to the 17th base at the 5' end of the target sequence (1≤n<17).

[0018] The design method or principle of the RT sequence and the PBS sequence can refer to the design method or principle related to the RT sequence and PBS sequence of pegRNA in the guide editing technology (Prime editing, PE) reported in the prior art.

[0019] The RT sequence may be in the form of an RT-S template or an RT-M template, preferably in the form of an RT-M template. The RT sequence in the form of the RT-S template contains only one target mutation site (i.e., the site where mutation is desired to occur), and a mutant base is introduced at the target mutation site. In addition to introducing a mutant base at the target mutation site, the RT sequence in the form of the RT-M template may also introduce a mutant base at other sites (remembered as an additional mutation site) other than the target mutation site, i.e., the mutant base introduced in the RT sequence may be the introduction of no less than two mutant bases at the target mutation site, or may be the introduction of an additional mutant base at other sites other than the target mutation site while introducing a mutant base at the target mutation site. The additional mutation site may be any site in the RT sequence other than the target mutation site. In practical applications, a suitable site may be selected as an additional mutation site and a suitable mutant base may be introduced according to actual needs, such as when only the base at the target mutation site is expected to cause a change in the amino acid sequence after mutation, and when the base at the additional mutation site does not cause a change in the amino acid sequence after mutation, the base at the additional mutation site may be designed to undergo a synonymous mutation. Further, the method of introducing the mutant base is base replacement. 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 mutant bases introduced at the target mutation site is specifically one. The number of additional mutant bases introduced at other sites other than the target mutation site may be one or two or more.

[0020] The linker sequence may be a linker sequence of any length. In a specific embodiment of the present invention, the linker sequence is a linker sequence of 8 bp in size, and is recorded as an 8-bp linker, and the 8-bp linker can be designed by pegLIT (https: / / doi.org / 10.1038 / s41587-021-01039-7) according to a method known in the art. In a specific embodiment of the present invention, the 8-bp linker is aacgagag, taaatatt, aaagaaga, atataatc, actctctg, cgaagagg, aagataac, aagtctta, ttataaga, aaggaagg or aattataa.

[0021] The nucleotide sequence of the tevopreQ1 motif is shown in Sequence 8.

[0022] In the above-mentioned complete system, the pegRNA is driven to express by a composite promoter; the composite promoter includes E35S promoter, CmYLCV promoter and OsU3 promoter in sequence.

[0023] In a specific embodiment of the present invention, the composite promoter is composed of E35S promoter, CmYLCV promoter and OsU3 promoter in sequence.

[0024] The nucleotide sequence of the E35S promoter is shown in SEQ ID NO: 10025-10461.

[0025] The nucleotide sequence of the CmYLCV promoter is shown in sequence 7 at positions 10462-10920.

[0026] The nucleotide sequence of the OsU3 promoter is shown in SEQ ID NO: 10932-11270.

[0027] In the above-mentioned complete system, the Cas9 nicking enzyme may be various Cas9n or variants thereof known in the prior art, including Cas9n derived from bacteria (such as SpCas9n, SaCas9n, SaCas9n-KKH, etc.), SpCas9 variant nicking enzymes recognizing different PAMs (such as xCas9n, Cas9n-NG, Cas9n-VQR, Cas9n-VRER, etc.), Cas9 high-fidelity enzyme variant nicking enzymes (such as HypaCas9n, eSpCas9(1.1)n, Cas9-HF1n, etc.), etc.

[0028] Further, the Cas9 nicking enzyme is Cas9maxn; the Cas9maxn is A1) or A2):

[0029] A1) The amino acid sequence is the protein shown in SEQ ID NO: 5;

[0030] A2) A protein having the same function as the amino acid sequence shown in SEQ ID NO: 5, wherein one or more amino acid residues are substituted and / or deleted and / or added.

[0031] Furthermore, the Cas9maxn is a1) or a2) or a3):

[0032] a1) the cDNA molecule or DNA molecule shown in positions 4422-8522 of SEQ ID NO: 7;

[0033] a2) a cDNA molecule or a DNA molecule that has 75% or more identity with the nucleotide sequence defined in a1) and encodes the Cas9maxn;

[0034] a3) a cDNA molecule or DNA molecule that hybridizes with the nucleotide sequence defined in b1) or b2) under stringent conditions and encodes the Cas9maxn.

[0035] The reverse transcriptase may be a reverse transcriptase derived from a virus, such as a reverse transcriptase derived from Moloney murine leukemia virus (M-MLV) or a reverse transcriptase derived from cauliflower mosaic virus (CaMV), or may be a reverse transcriptase derived from a virus in bacteria, such as a reverse transcriptase derived from Escherichia coli.

[0036] Further, the reverse transcriptase is M-MLV RT; the M-MLV RT is B1) or B2):

[0037] B1) The amino acid sequence is the protein shown in SEQ ID NO: 1;

[0038] B2) A protein having the same function as the amino acid sequence shown in SEQ ID NO: 1, wherein one or more amino acid residues are substituted and / or deleted and / or added.

[0039] Furthermore, the coding gene of the M-MLV RT is b1) or b2) or b3):

[0040] b1) the cDNA molecule or DNA molecule shown in positions 2292-4322 of SEQ ID NO: 7;

[0041] b2) a cDNA molecule or a DNA molecule that has 75% or more identity with the nucleotide sequence defined in b1) and encodes the M-MLV RT;

[0042] b3) a cDNA molecule or a DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in b1) or b2) and encodes the M-MLV RT.

[0043] The self-cleaving oligopeptide can be a 2A self-cleaving oligopeptide derived from a viral genome, such as foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis virus type A (ERAV) (E2A) peptide, Thosea asigna virus (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, Theiler virus 2A peptide and encephalomyocarditis virus 2A peptide.

[0044] Further, the 2A self-cleaving oligopeptide derived from the viral genome is a 2A self-cleaving oligopeptide derived from porcine teschovirus-1; the amino acid sequence of the 2A self-cleaving oligopeptide (P2A) derived from porcine teschovirus-1 is C1) or C2):

[0045] C1) The amino acid sequence is the protein shown in SEQ ID NO: 3;

[0046] C2) A protein having the same function as the amino acid sequence shown in SEQ ID NO: 3, wherein one or more amino acid residues are substituted and / or deleted and / or added.

[0047] The coding gene of P2A is c1) or c2) or c3):

[0048] c1) the cDNA molecule or DNA molecule shown in positions 8673-8729 of SEQ ID NO: 7;

[0049] c2) a cDNA molecule or a DNA molecule that has 75% or more identity with the nucleotide sequence defined in c1) and encodes the P2A;

[0050] c3) a cDNA molecule or a DNA molecule that hybridizes with the nucleotide sequence defined in c1) or c2) under stringent conditions and encodes the P2A.

[0051] The screening agent resistance protein can be various resistance proteins known in the prior art, such as kanamycin resistance protein, herbicide resistance protein (such as glyphosate resistance protein, glufosinate resistance protein), phosphomannose isomerase, etc.

[0052] Further, the screening agent resistance protein is hygromycin phosphotransferase; the hygromycin phosphotransferase is D1) or D2):

[0053] D1) The amino acid sequence is the protein shown in SEQ ID NO: 4;

[0054] D2) A protein having the same function as the amino acid sequence shown in SEQ ID NO: 4, wherein one or several amino acid residues are substituted and / or deleted and / or added.

[0055] Furthermore, the coding gene of the hygromycin phosphotransferase is d1) or d2) or d3):

[0056] d1) the cDNA molecule or DNA molecule shown in positions 8730-9755 of SEQ ID NO: 7;

[0057] d2) a cDNA molecule or a DNA molecule that has 75% or more identity with the nucleotide sequence defined in d1) and encodes the hygromycin phosphotransferase;

[0058] d3) a cDNA molecule or a DNA molecule which hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the hygromycin phosphotransferase.

[0059] Another object of the present invention is to provide new uses for the above-mentioned complete system.

[0060] The present invention provides the application of the above-mentioned complete set of systems in any of the following S1)-S4):

[0061] S1) Editing of genome sequences of organisms or biological cells;

[0062] S2) preparing an edited product of an organism or an organism cell genome sequence;

[0063] S3) improving the editing efficiency of the genome sequence of an organism or biological cell;

[0064] S4) Prepare products that improve the editing efficiency of the genome sequence of an organism or biological cell.

[0065] Another object of the present invention is to provide any of the following methods T1)-T3):

[0066] T1) A method for editing a genome sequence, comprising the following steps: causing an organism or biological cell to express the fusion protein, the esgRNA and the pegRNA;

[0067] T2) A method for improving the editing efficiency of a genome sequence of an organism or biological cell, comprising the following steps: causing the organism or biological cell to express the above-mentioned fusion protein, the above-mentioned esgRNA and the above-mentioned pegRNA;

[0068] T3) A method for preparing a biological mutant, comprising the following steps: editing the genome sequence of an organism or a biological cell according to the method described in T1) or T2) to obtain a biological mutant.

[0069] In the above method, in T1) and T2), the method for making the organism or biological cell express the above fusion protein, the above esgRNA and the above pegRNA is to introduce the coding gene of the above fusion protein, the DNA molecule that transcribes the above esgRNA and the DNA molecule that transcribes the above pegRNA into the target plant.

[0070] Further, in T1) and T2), the coding gene of the fusion protein, the DNA molecule that transcribes the esgRNA, and the DNA molecule that transcribes the pegRNA are introduced into the target plant through a recombinant expression vector. The coding gene of the fusion protein, the DNA molecule that transcribes the esgRNA, and the DNA molecule that transcribes the pegRNA can be introduced into the target plant through the same recombinant expression vector, or can be introduced into the target plant through two or more recombinant expression vectors.

[0071] In a specific embodiment of the present invention, the coding gene of the fusion protein, the DNA molecule that transcribes the above esgRNA, and the DNA molecule that transcribes the above pegRNA are introduced into the target plant through the same recombinant expression vector. The recombinant expression vector includes an expression cassette composed of a promoter, a coding gene of M-MLV RT, a coding gene of Cas9maxn, a coding gene of a self-cleaving oligopeptide P2A, a coding gene of a screening agent resistance protein HPT, and a terminator, an expression cassette composed of a promoter, a DNA molecule that transcribes esgRNA, and poly T, and an expression cassette composed of a promoter, a DNA molecule that transcribes pegRNA, and poly T. The recombinant expression vector is specifically PE-P6 below. 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.

[0072] In any of the above-mentioned complete systems, applications or methods, the editing of the genome sequence includes base substitution (such as single base substitution and multiple base substitution), base insertion (such as single base insertion and multiple base insertion) and base deletion (such as single base deletion and multiple base deletion) of the genome sequence. In a specific embodiment of the present invention, the editing of the genome sequence is base substitution of the genome sequence.

[0073] In any of the above-mentioned complete systems, applications or methods, the organism is X1) or X2) or X3) or X4):

[0074] X1) Plants or animals;

[0075] X2) monocots or dicots;

[0076] X3) Gramineae;

[0077] X4) Rice.

[0078] The biological cell is Y1) or Y2) or Y3) or Y4):

[0079] Y1) plant cells or animal cells;

[0080] Y2) a monocotyledonous plant cell or a dicotyledonous plant cell;

[0081] Y3) Gramineae plant cells;

[0082] Y4) Rice cells.

[0083] In order to further improve the editing efficiency of the guide editing system, the present invention knocked out the rice MMR repair gene based on the PE-P6 guide editing system to obtain the guide editing system PE-P6 ΔOsMLH1 RT-S and Guided Editing System PE-P6 ΔOsMLH1 Compared with the PE-P6 guide editing system, after knocking out the OsMLH1 gene, the guide editing system PE-P6 ΔOsMLH1 RT-S and PE-P6 ΔOsMLH1 RT-M can further improve the editing efficiency of the target; and guide the editing system PE-P6 ΔOsMLH1 RT-M can maximize the editing efficiency of rice targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 For boot editing systems 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 diagram of the structure of the RT-M expression vector.

[0085] Figure 2 Schematic diagram of the RT-M template format.

[0086] Figure 3 To guide the editing efficiency of rice callus at 8 targets in the editing systems PE-P3, PE-P4, PE-P5, PE-P6, and PE-P7. DETAILED DESCRIPTION

[0087] The present invention is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, instruments, etc. used in the following examples are all commercially available unless otherwise specified. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0088] In the following examples, the editing efficiency of the guide editing system in rice callus = (the number of reads with all mutation sites detected in group 1 / the total number of reads × 100% + the number of reads with all mutation sites detected in group 2 / the total number of reads × 100% + the number of reads with all mutation sites detected in group 3 / the total number of reads × 100% + the number of reads with all mutation sites detected in group 4 / the total number of reads × 100%) / 4.

[0089] The editing efficiency of the guide editing system in rice T0 seedlings = the number of positive T0 seedlings with mutations at all mutation sites / the total number of positive T0 seedlings analyzed × 100%.

[0090] Nipponbare rice: References: Liang Weihong, Wang Gaohua, Du Jingyao, et al. Effects of sodium nitroprusside and its photolysis products on the growth of Nipponbare rice seedlings and the expression of five hormone marker genes [J]. Journal of Henan Normal University (Nature Edition), 2017(2):48-52.; Available to the public from Beijing Academy of Agricultural and Forestry Sciences.

[0091] Recovery medium: N6 solid medium containing 200 mg / L Timentin.

[0092] Screening medium: N6 solid medium containing 50 mg / L hygromycin.

[0093] Differentiation medium: N6 solid medium containing 2 mg / L KT, 0.2 mg / L NAA, 0.5 g / L glutamic acid, and 0.5 g / L proline.

[0094] Rooting medium: N6 solid medium containing 0.2 mg / L NAA, 0.5 g / L glutamic acid, and 0.5 g / L proline.

[0095] Example 1. Design of main components of different guide editing systems and their expression vectors

[0096] 1. Design of main components of different boot editing systems

[0097] The guide editing system PE-P3 includes fusion protein, esgRNA and pegRNA; the fusion protein includes Cas9 nicking enzyme (such as Cas9n (H840A)), reverse transcriptase (such as M-MLV), self-cleaving oligopeptide (such as P2A) and screening marker protein (such as HPT); esgRNA is used to produce non-editing chain nicks; pegRNA is composed of esgRNA, reverse transcription template sequence (RT sequence) and primer binding site sequence (PBS sequence) in sequence.

[0098] The guide editing system PE-P4 includes fusion protein and pegRNA; the fusion protein includes Cas9 nicking enzyme (such as Cas9n (H840A)), reverse transcriptase (such as M-MLV), self-cleaving oligopeptide (such as P2A) and screening marker protein (such as HPT); pegRNA is composed of esgRNA, reverse transcription template sequence (RT sequence) and primer binding site sequence (PBS sequence) in sequence.

[0099] The guide editing system PE-P5 includes fusion protein and pegRNA; the fusion protein includes Cas9 nicking enzyme (such as R221K with improved cutting efficiency, Cas9n with N394K mutations (Cas9maxn)), reverse transcriptase (such as M-MLV), self-cleaving oligopeptide (such as P2A) and screening marker protein (such as HPT); pegRNA is composed of esgRNA, reverse transcription template sequence (RT sequence) and primer binding site sequence (PBS sequence) in sequence, and the composite promoter system (E35S+CmYLCV+OsU3) drives pegRNA expression.

[0100] The guide editing system PE-P6 includes fusion protein and pegRNA; the fusion protein includes Cas9 nicking enzyme (such as Cas9n (Cas9maxn) with R221K and N394K mutations with improved cutting efficiency), reverse transcriptase (such as M-MLV), self-cleaving oligopeptide (such as P2A) and screening marker protein (such as HPT); pegRNA is composed of esgRNA, reverse transcription template sequence (RT sequence) and primer binding site sequence (PBS sequence) in sequence, and an 8-bp linker and tevopreQ1 motif are introduced at the 3' end of pegRNA (to form engineered pegRNA (epegRNA) to further enhance the stability of the 3' end of pegRNA and prevent it from being degraded), and a composite promoter system (E35S+CmYLCV+OsU3) drives pegRNA expression.

[0101] The guide editing system PE-P7 includes fusion protein and pegRNA; the fusion protein includes Cas9 nicking enzyme (such as Cas9n (Cas9maxn) with R221K and N394K mutations with improved cutting efficiency), reverse transcriptase (such as M-MLV), self-cleaving oligopeptide (such as P2A) and screening marker protein (such as HPT); pegRNA is composed of esgRNA, reverse transcription template sequence (RT sequence) and primer binding site sequence (PBS sequence) in sequence, and an 8-bp linker and mpknot motif are introduced at the 3' end of pegRNA (to form engineered pegRNA (epegRNA) to further enhance the stability of the 3' end of pegRNA and prevent it from being degraded), and a composite promoter system (E35S+CmYLCV+OsU3) drives pegRNA expression.

[0102] Boot Editing System PE-P6 OE hMLH1dn RT-S includes fusion protein, hMLH1dn and pegRNA; the fusion protein includes Cas9 nicking enzyme (such as Cas9n (Cas9maxn) with R221K and N394K mutations with improved cutting efficiency), reverse transcriptase (such as M-MLV), self-cleaving oligopeptide (such as P2A) and screening marker protein (such as HPT); pegRNA is composed of esgRNA, reverse transcription template sequence (RT sequence) and primer binding site sequence (PBS sequence) in sequence, the RT sequence is in the form of RT-S template (only the mutant base is introduced at the target mutation site, and all mutation sites are only the target mutation site), and an 8-bp linker and tevopreQ1 motif are introduced at the 3' end of pegRNA (to form an engineered pegRNA (epegRNA) to further enhance the stability of the 3' end of pegRNA and prevent it from being degraded), and a composite promoter system (E35S+CmYLCV+OsU3) drives pegRNA expression.

[0103] Boot Editing System PE-P6 OE hMLH1dnRT-M includes fusion protein, hMLH1dn and pegRNA; the fusion protein includes Cas9 nicking enzyme (such as Cas9n (Cas9maxn) with R221K and N394K mutations with improved cleavage efficiency), reverse transcriptase (such as M-MLV), self-cleaving oligopeptide (such as P2A) and screening marker protein (such as HPT); pegRNA is composed of esgRNA, reverse transcription template sequence (RT sequence) and primer binding site sequence (PBS sequence) in sequence, and the RT sequence is in the form of RT-M template (in addition to introducing mutant bases at the target mutation site, additional mutant bases are introduced at other sites (additional mutation sites) other than the target mutation site, and all mutation sites are composed of target mutation sites and additional mutation sites), and 8-bplinker and tevopreQ1 motifs are introduced at the 3' end of pegRNA (to form engineered pegRNA (epegRNA) to further enhance pegRNA The stability of the 3' end prevents it from being degraded), and the composite promoter system (E35S+CmYLCV+OsU3) drives the expression of pegRNA.

[0104] Boot Editing System PE-P6 ΔOsMLH1 RT-S includes fusion protein, esgRNA and pegRNA; the fusion protein includes Cas9 nicking enzyme (such as Cas9n (Cas9maxn) with R221K and N394K mutations with improved cleavage efficiency), reverse transcriptase (such as M-MLV), self-cleaving oligopeptide (such as P2A) and selection marker protein (such as HPT); esgRNA contains a target for knocking out rice MLH1 gene (MLH1 gene sequence is shown in sequence 14) (knocking out rice MMR repair gene to prevent intracellular MMR repair); pegRNA is composed of esgRNA, reverse transcription template sequence (RT sequence) and primer binding site sequence (PBS sequence) in sequence, the RT sequence is in the form of RT-S template (mutated bases are introduced only at the target mutation site, and all mutation sites are only target mutation sites), and 8-bp linker and tevopreQ1 motif are introduced at the 3' end of pegRNA (to form engineered pegRNA (epegRNA) to further enhance pegRNA The stability of the 3' end prevents it from being degraded), and the composite promoter system (E35S+CmYLCV+OsU3) drives the expression of pegRNA.

[0105] Boot Editing System PE-P6 ΔOsMLH1RT-M includes fusion protein, esgRNA and pegRNA; the fusion protein includes Cas9 nicking enzyme (such as Cas9n (Cas9maxn) with R221K and N394K mutations with improved cutting efficiency), reverse transcriptase (such as M-MLV), self-cleaving oligopeptide (such as P2A) and selection marker protein (such as HPT); esgRNA contains a target for knocking out the rice MLH1 gene (knocking out the rice MMR repair gene to prevent the intracellular MMR repair effect); pegRNA is composed of esgRNA, reverse transcription template sequence (RT sequence) and primer binding site sequence (PBS sequence) in sequence, the RT sequence is in the form of RT-M template (in addition to introducing mutant bases at the target mutation site, additional mutant bases are introduced at other sites (additional mutation sites) other than the target mutation site, and all mutation sites are composed of target mutation sites and additional mutation sites), and 8-bplinker and tevopreQ1 motifs are introduced at the 3' end of pegRNA (to form an engineered pegRNA (epegRNA) to further enhance the pegRNA The stability of the 3' end prevents it from being degraded), and the composite promoter system (E35S+CmYLCV+OsU3) drives the expression of pegRNA.

[0106] The schematic diagram of RT-M in the epegRNA involved in the above-mentioned guide editing system is as follows Figure 2 As shown, red indicates the target mutation site and blue indicates the additional mutation site.

[0107] The 8-bp linker in the epegRNA involved in the above-mentioned guide editing system was designed by pegLIT (https: / / doi.org / 10.1038 / s41587-021-01039-7).

[0108] 2. Expression vector design for different guide editing systems

[0109] The schematic diagrams of the structures of the above-mentioned guide editing system expression vectors are shown in Figure 1 shown.

[0110] The expression vector of the guide editing system PE-P3 includes the M-MLV&Cas9n(H840A)&HPT expression cassette, the esgRNA expression cassette and the pegRNA expression cassette. In the M-MLV&Cas9n(H840A)&HPT expression cassette, M-MLV is fused to the N-terminus of Cas9n(H840A), and the screening agent resistance protein is fused to the C-terminus of Cas9n(H840A) through the self-cleaving polypeptide P2A. The expression cassette is driven by the ZmUbi1 promoter. The esgRNA expression cassette is driven by the OsU6a promoter. The pegRNA expression cassette is driven by the OsU3 promoter.

[0111] The expression vector of the guide editing system PE-P4 includes an M-MLV&Cas9n(H840A)&HPT expression cassette and a pegRNA expression cassette. In the M-MLV&Cas9n(H840A)&HPT expression cassette, M-MLV is fused to the N-terminus of Cas9n(H840A), and the screening agent resistance protein is fused to the C-terminus of Cas9n(H840A) through the self-cleaving polypeptide P2A. The expression cassette is driven by the ZmUbi1 promoter. The pegRNA expression cassette is driven by the OsU3 promoter.

[0112] The expression vector of the guide editing system PE-P5 includes an M-MLV&Cas9maxn&HPT expression cassette and a pegRNA expression cassette. In the M-MLV&Cas9maxn&HPT expression cassette, M-MLV is fused to the N-terminus of Cas9maxn, and the selection agent resistance protein is fused to the C-terminus of Cas9maxn through the self-cleaving polypeptide P2A. The expression cassette is driven by the ZmUbi1 promoter. The pegRNA expression cassette is driven by the composite promoter E35S+CmYLCV+OsU3.

[0113] The expression vector of the guide editing system PE-P6 includes an M-MLV&Cas9maxn&HPT expression cassette and a pegRNA expression cassette. In the M-MLV&Cas9maxn&HPT expression cassette, M-MLV is fused to the N-terminus of Cas9maxn, and the selection agent resistance protein is fused to the C-terminus of Cas9maxn through the self-cleaving polypeptide P2A. The expression cassette is driven by the ZmUbi1 promoter. In the pegRNA expression cassette, an 8-bp linker and a tevopreQ1 motif are introduced at the 3' end of the pegRNA, and the expression cassette is driven by the composite promoter E35S+CmYLCV+OsU3.

[0114] The expression vector of the guide editing system PE-P7 includes an M-MLV&Cas9maxn&HPT expression cassette and a pegRNA expression cassette. In the M-MLV&Cas9maxn&HPT expression cassette, M-MLV is fused to the N-terminus of Cas9maxn, and the selection agent resistance protein is fused to the C-terminus of Cas9maxn through the self-cleaving polypeptide P2A. The expression cassette is driven by the ZmUbi1 promoter. In the pegRNA expression cassette, an 8-bp linker and tmpknot motif are introduced at the 3' end of the pegRNA, and the expression cassette is driven by the composite promoter E35S+CmYLCV+OsU3.

[0115] Boot Editing System PE-P6 OE hMLH1dnThe expression vector of RT-S includes M-MLV&Cas9maxn&HPT expression cassette, hMLH1dn expression cassette and pegRNA expression cassette. In the M-MLV&Cas9maxn&HPT expression cassette, M-MLV is fused to the N-terminus of Cas9maxn, and the screening agent resistance protein is fused to the C-terminus of Cas9maxn through the self-cleaving polypeptide P2A. The expression cassette is driven by the ZmUbi1 promoter. The hMLH1dn expression cassette is driven by the OsActin promoter. The RT sequence in the pegRNA expression cassette is in the form of an RT-S template, and an 8-bp linker and tevopreQ1 motif are introduced at the 3' end of the pegRNA. The expression cassette is driven by the composite promoter E35S+CmYLCV+OsU3.

[0116] Boot Editing System PE-P6 OE hMLH1dn The expression vector of RT-M includes M-MLV&Cas9maxn&HPT expression cassette, hMLH1dn expression cassette and pegRNA expression cassette. In the M-MLV&Cas9maxn&HPT expression cassette, M-MLV is fused to the N-terminus of Cas9maxn, and the screening agent resistance protein is fused to the C-terminus of Cas9maxn through the self-cleaving polypeptide P2A. The expression cassette is driven by the ZmUbi1 promoter. The hMLH1dn expression cassette is driven by the OsActin promoter. The RT sequence in the pegRNA expression cassette is in the form of an RT-M template, and an 8-bp linker and tevopreQ1 motif are introduced at the 3' end of the pegRNA. The expression cassette is driven by the composite promoter E35S+CmYLCV+OsU3.

[0117] Boot Editing System PE-P6 ΔOsMLH1 The expression vector of RT-S includes M-MLV&Cas9maxn&HPT expression cassette, esgRNA expression cassette and pegRNA expression cassette. In the M-MLV&Cas9maxn&HPT expression cassette, M-MLV is fused to the N-terminus of Cas9maxn, and the screening agent resistance protein is fused to the C-terminus of Cas9maxn through the self-cleaving polypeptide P2A. The expression cassette is driven by the ZmUbi1 promoter. The esgRNA expression cassette contains a target for knocking out the rice MLH1 gene (sequence 13), and the expression cassette is driven by OsU6a. The RT sequence in the pegRNA expression cassette is in the form of an RT-S template, and an 8-bp linker and tevopreQ1 motif are introduced at the 3' end of the pegRNA. The expression cassette is driven by the composite promoter E35S+CmYLCV+OsU3.

[0118] Boot Editing System PE-P6 ΔOsMLH1The expression vector of RT-M includes M-MLV&Cas9maxn&HPT expression cassette, esgRNA expression cassette and pegRNA expression cassette. In the M-MLV&Cas9maxn&HPT expression cassette, M-MLV is fused to the N-terminus of Cas9maxn, and the screening agent resistance protein is fused to the C-terminus of Cas9maxn through the self-cleaving polypeptide P2A. The expression cassette is driven by the ZmUbi1 promoter. The esgRNA expression cassette contains a target site for knocking out the rice MLH1 gene, and the expression cassette is driven by OsU6a. The RT sequence in the pegRNA expression cassette is in the form of an RT-M template, and an 8-bp linker and tevopreQ1 motif are introduced at the 3' end of the pegRNA. The expression cassette is driven by the composite promoter E35S+CmYLCV+OsU3.

[0119] Example 2: Construction of expression vectors of different guide editing systems and comparison of their efficiency in base editing of rice genome

[0120] 1. Construction of expression vectors of different guide editing systems

[0121] The following recombinant vectors were artificially constructed, each of which is a circular plasmid:

[0122] There are a total of 8 expression vectors of the guide editing system PE-P3, namely 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.

[0123] There are a total of 8 expression vectors of the guide editing system PE-P4, namely 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 vectors.

[0124] There are a total of 8 expression vectors of the guide editing system PE-P5, namely 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 vectors.

[0125] There are a total of 8 expression vectors of the guide editing system PE-P6, namely 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 vectors.

[0126] There are a total of 8 expression vectors of the guide editing system PE-P7, namely 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 vectors.

[0127] Boot Editing System PE-P6 OE hMLH1dn There are three expression vectors for RT-S, namely PE-P6 OE hMLHdn RT-S-OsEPSPS-2, PE-P6 OE hMLHdn RT-S-OsGRF4-T2, PE-P6 OE hMLHdn RT-S-OsSD1 vector.

[0128] Boot Editing System PE-P6 OE hMLH1dn There are three expression vectors for RT-M, namely PE-P6 OE hMLHdn RT-M-OsEPSPS-2, PE-P6 OE hMLHdn RT-M-OsGRF4-T2, PE-P6 OE hMLHdn RT-M-OsSD1 vector.

[0129] Boot Editing System PE-P6 ΔOsMLH1 There are three expression vectors for RT-S, namely PE-P6 ΔOsMLH1 RT-S-OsEPSPS-2, PE-P6 ΔOsMLH1 RT-S-OsGRF4-T2, PE-P6 ΔOsMLH1 RT-S-OsSD1 vector.

[0130] Boot Editing System PE-P6 ΔOsMLH1 There are three expression vectors for RT-M, namely PE-P6 ΔOsMLH1 RT-M-OsEPSPS-2, PE-P6 ΔOsMLH1 RT-M-OsGRF4-T2, PE-P6 ΔOsMLH1 RT-M-OsSD1 vector.

[0131] The nucleotide sequence of the PE-P3-RTM-OsALS-3 recombinant expression vector (denoted as PE-P3-1 vector) is shown in Sequence 6. Among them, the nucleotide sequence of the 104th to 2075th position of Sequence 6 is the nucleotide sequence of the ZmUbi1 promoter; the 2292th to 4322nd position is the coding sequence of the M-MLV RT protein, which encodes the M-MLV shown in Sequence 1. RT protein; positions 4422-8522 are the coding sequence of Cas9n (H840A) protein (excluding the stop codon), encoding the Cas9n (H840A) protein shown in sequence 2; positions 8673-8729 are the coding sequence of P2A, encoding the protein shown in sequence 3; positions 8730-9755 are the coding sequence of hygromycin phosphotransferase (HPT), encoding the HPT protein shown in sequence 4; positions 9762-10016 are the Nos terminator sequence; positions 10025-10490 are the nucleotide sequence of the OsU6a promoter, positions 10491-10510 are the esgRNA target sequence for generating a non-coding strand nick, positions 10511-10596 are the esgRNA backbone sequence for generating a non-coding strand nick, and positions 10597-10605 are Poly T; positions 10606-10986 are the nucleotide sequence of the OsU3 promoter, positions 10987-11006 are the pegRNA-01 target sequence, positions 11007-11092 are the esgRNA backbone sequence corresponding to pegRNA-01, positions 11093-11119 are the RT&PBS sequence on pegRNA-01, and positions 11120-11127 are Poly T. The esgRNA target sequence for generating the non-coding strand nick corresponding to pegRNA-01 in the PE-P3-RTM-OsALS-3 recombinant expression vector, the pegRNA-01 target sequence, and the RT&PBS sequence on pegRNA-01 are shown in Table 1.

[0132] The nucleotide sequence of the PE-P3-RTM-OsEPSPS-2 recombinant expression vector (referred to as the PE-P3-2 vector) is obtained by replacing the esgRNA target sequence that produces a non-coding chain nick, the pegRNA-01 target sequence, and the RT&PBS sequence on pegRNA-01 corresponding to pegRNA-01 in the PE-P3-1 vector with the esgRNA target sequence that produces a non-coding chain nick, the pegRNA-02 target sequence, and the RT&PBS sequence on pegRNA-02 corresponding to pegRNA-02, respectively, while keeping other sequences unchanged.

[0133] The nucleotide sequence of the PE-P3-RTM-OsGRF4-T2 recombinant expression vector (referred to as PE-P3-3 vector) is obtained by replacing the esgRNA target sequence that produces a non-coding chain nick, the pegRNA-01 target sequence, and the RT&PBS sequence on pegRNA-01 corresponding to pegRNA-01 in the PE-P3-1 vector with the esgRNA target sequence that produces a non-coding chain nick, the pegRNA-03 target sequence, and the RT&PBS sequence on pegRNA-03 corresponding to pegRNA-03, respectively, while keeping other sequences unchanged.

[0134] The nucleotide sequence of the PE-P3-RTM-OsSD1 recombinant expression vector (referred to as PE-P3-4 vector) is obtained by replacing the esgRNA target sequence that produces a non-coding chain nick, the pegRNA-01 target sequence, and the RT&PBS sequence on pegRNA-01 corresponding to pegRNA-01 in the PE-P3-1 vector with the esgRNA target sequence that produces a non-coding chain nick, the pegRNA-04 target sequence, and the RT&PBS sequence on pegRNA-04 corresponding to pegRNA-04, respectively, while keeping other sequences unchanged.

[0135] The nucleotide sequence of the PE-P3-RTM-OsCold1 recombinant expression vector (referred to as PE-P3-5 vector) is obtained by replacing the esgRNA target sequence that produces a non-coding chain nick, the pegRNA-01 target sequence, and the RT&PBS sequence on pegRNA-01 corresponding to pegRNA-01 in the PE-P3-1 vector with the esgRNA target sequence that produces a non-coding chain nick, the pegRNA-05 target sequence, and the RT&PBS sequence on pegRNA-05 corresponding to pegRNA-05, respectively, while keeping other sequences unchanged.

[0136] The nucleotide sequence of the PE-P3-RTM-OsALS-1 recombinant expression vector (referred to as PE-P3-6 vector) is obtained by replacing the esgRNA target sequence that produces a non-coding chain nick, the pegRNA-01 target sequence, and the RT&PBS sequence on pegRNA-01 corresponding to pegRNA-01 in the PE-P3-1 vector with the esgRNA target sequence that produces a non-coding chain nick, the pegRNA-06 target sequence, and the RT&PBS sequence on pegRNA-06 corresponding to pegRNA-06, respectively, while keeping other sequences unchanged.

[0137] The nucleotide sequence of the PE-P3-RTM-OsACC-1 recombinant expression vector (referred to as the PE-P3-7 vector) is obtained by replacing the esgRNA target sequence that produces a non-coding chain nick, the pegRNA-01 target sequence, and the RT&PBS sequence on pegRNA-01 corresponding to pegRNA-01 in the PE-P3-1 vector with the esgRNA target sequence that produces a non-coding chain nick, the pegRNA-07 target sequence, and the RT&PBS sequence on pegRNA-07 corresponding to pegRNA-07, respectively, while keeping other sequences unchanged.

[0138] The nucleotide sequence of the PE-P3-RTM-OsGS3 recombinant expression vector (referred to as the PE-P3-8 vector) is obtained by replacing the esgRNA target sequence that produces a non-coding chain incision, the pegRNA-01 target sequence, and the RT&PBS sequence on pegRNA-01 corresponding to pegRNA-01 in the PE-P3-1 vector with the esgRNA target sequence that produces a non-coding chain incision, the pegRNA-08 target sequence, and the RT&PBS sequence on pegRNA-08 corresponding to pegRNA-08, respectively, while keeping other sequences unchanged.

[0139] The nucleotide sequence of the PE-P4-RTM-OsALS-3 recombinant expression vector (denoted as PE-P4-1 vector) is the sequence obtained by deleting the nucleotide sequence of the OsU6a promoter, the esgRNA target sequence that produces a non-coding strand nick, the esgRNA backbone sequence that produces a non-coding strand nick, and the Poly T sequence at positions 10597-10605 in the PE-P3-1 vector, while keeping other sequences unchanged.

[0140] The nucleotide sequence of the PE-P4-RTM-OsEPSPS-2 recombinant expression vector (referred to as the PE-P4-2 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-02 target sequence and the RT&PBS sequence on the pegRNA-02, respectively, while keeping other sequences unchanged.

[0141] The nucleotide sequence of the PE-P4-RTM-OsGRF4-T2 recombinant expression vector (referred to as PE-P4-3 vector) is obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P4-1 vector with the pegRNA-03 target sequence and the RT&PBS sequence on pegRNA-03, respectively, while keeping other sequences unchanged.

[0142] The nucleotide sequence of the PE-P4-RTM-OsSD1 recombinant expression vector (referred to as PE-P4-4 vector) is obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P4-1 vector with the pegRNA-04 target sequence and the RT&PBS sequence on pegRNA-04, respectively, while keeping other sequences unchanged.

[0143] The nucleotide sequence of the PE-P4-RTM-OsCold1 recombinant expression vector (referred to as PE-P4-5 vector) is obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P4-1 vector with the pegRNA-05 target sequence and the RT&PBS sequence on pegRNA-05, respectively, while keeping other sequences unchanged.

[0144] The nucleotide sequence of the PE-P4-RTM-OsALS-1 recombinant expression vector (referred to as PE-P4-6 vector) is the sequence obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P4-1 vector with the pegRNA-06 target sequence and the RT&PBS sequence on pegRNA-06, respectively, while keeping other sequences unchanged.

[0145] The nucleotide sequence of the PE-P4-RTM-OsACC-1 recombinant expression vector (denoted as PE-P4-7 vector) is the sequence obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P4-1 vector with the pegRNA-07 target sequence and the RT&PBS sequence on pegRNA-07, respectively, while keeping other sequences unchanged.

[0146] The nucleotide sequence of the PE-P4-RTM-OsGS3 recombinant expression vector (denoted as PE-P4-8 vector) is the sequence obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P4-1 vector with the pegRNA-08 target sequence and the RT&PBS sequence on pegRNA-08, respectively, while keeping other sequences unchanged.

[0147] The nucleotide sequence of the PE-P5-RTM-OsALS-3 recombinant expression vector (denoted as PE-P5-1 vector) is shown in SEQ ID NO: 7. Among them, the nucleotide sequence of the 104th to 2075th positions of SEQ ID NO: 7 is the nucleotide sequence of the ZmUbi1 promoter; the 2292th to 4322nd positions are the coding sequence of the M-MLV RT protein, which encodes the M-MLV shown in SEQ ID NO: 1. RT protein; positions 4422-8522 are the coding sequence of Cas9maxn protein (excluding the stop codon), encoding the Cas9maxn protein shown in sequence 5; positions 8673-8729 are the coding sequence of P2A, encoding the protein shown in sequence 3; positions 8730-9755 are the coding sequence of HPT, encoding the HPT protein shown in sequence 4; positions 9762-10016 are the Nos terminator sequence; positions 10025-10461 are the nucleotide sequence of the E35s promoter; positions 10462-10920 are the nucleotide sequence of the CmYLCV promoter; 0932-11270 is the nucleotide sequence of the OsU3 promoter; positions 11271-11347 are the nucleotide sequence of tRNA; positions 11348-11367 are the pegRNA-01 target sequence, positions 11368-11453 are the esgRNA backbone sequence corresponding to pegRNA-01, positions 11454-11478 are the RT&PBS sequences on pegRNA-01, positions 11479-11546 are the nucleotide sequence of HDV (HDV is used in combination with tRNA and can be used to cut sgRNA); positions 11547-11554 are Poly T; positions 11555-11825 are the HSP terminator sequence.

[0148] The nucleotide sequence of the PE-P5-RTM-OsEPSPS-2 recombinant expression vector (referred to as the PE-P5-2 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-02 target sequence and the RT&PBS sequence on the pegRNA-02, respectively, while keeping other sequences unchanged.

[0149] The nucleotide sequence of the PE-P5-RTM-OsGRF4-T2 recombinant expression vector (referred to as PE-P5-3 vector) is obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P5-1 vector with the pegRNA-03 target sequence and the RT&PBS sequence on pegRNA-03, respectively, while keeping other sequences unchanged.

[0150] The nucleotide sequence of the PE-P5-RTM-OsSD1 recombinant expression vector (referred to as PE-P5-4 vector) is obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P5-1 vector with the pegRNA-04 target sequence and the RT&PBS sequence on pegRNA-04, respectively, while keeping other sequences unchanged.

[0151] The nucleotide sequence of the PE-P5-RTM-OsCold1 recombinant expression vector (referred to as PE-P5-5 vector) is obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P5-1 vector with the pegRNA-05 target sequence and the RT&PBS sequence on pegRNA-05, respectively, while keeping other sequences unchanged.

[0152] The nucleotide sequence of the PE-P5-RTM-OsALS-1 recombinant expression vector (referred to as PE-P5-6 vector) is the sequence obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P5-1 vector with the pegRNA-06 target sequence and the RT&PBS sequence on pegRNA-06, respectively, while keeping other sequences unchanged.

[0153] The nucleotide sequence of the PE-P5-RTM-OsACC-1 recombinant expression vector (denoted as PE-P5-7 vector) is the sequence obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P5-1 vector with the pegRNA-07 target sequence and the RT&PBS sequence on pegRNA-07, respectively, while keeping other sequences unchanged.

[0154] The nucleotide sequence of the PE-P5-RTM-OsGS3 recombinant expression vector (referred to as PE-P5-8 vector) is obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P5-1 vector with the pegRNA-08 target sequence and the RT&PBS sequence on pegRNA-08, respectively, while keeping other sequences unchanged.

[0155] The nucleotide sequence of the PE-P6-RTM-OsALS-3 recombinant expression vector (denoted as PE-P6-1 vector) is obtained by inserting the 8-bp linker and tevopreQ1 motif (sequence 8) of the pegRNA-01 target between the RT&PBS sequence and the HDV sequence of the PE-P5-1 vector, while keeping the other sequences unchanged.

[0156] The nucleotide sequence of the PE-P6-RTM-OsEPSPS-2 recombinant expression vector (referred to as the PE-P6-2 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-02 target sequence and the RT&PBS sequence on the pegRNA-02, respectively, while keeping other sequences unchanged.

[0157] The nucleotide sequence of the PE-P6-RTM-OsGRF4-T2 recombinant expression vector (referred to as PE-P6-3 vector) is obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P6-1 vector with the pegRNA-03 target sequence and the RT&PBS sequence on pegRNA-03, respectively, while keeping other sequences unchanged.

[0158] The nucleotide sequence of the PE-P6-RTM-OsSD1 recombinant expression vector (referred to as PE-P6-4 vector) is obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P6-1 vector with the pegRNA-04 target sequence and the RT&PBS sequence on pegRNA-04, respectively, while keeping other sequences unchanged.

[0159] The nucleotide sequence of the PE-P6-RTM-OsCold1 recombinant expression vector (referred to as PE-P6-5 vector) is obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P6-1 vector with the pegRNA-05 target sequence and the RT&PBS sequence on pegRNA-05, respectively, while keeping other sequences unchanged.

[0160] The nucleotide sequence of the PE-P6-RTM-OsALS-1 recombinant expression vector (referred to as PE-P6-6 vector) is the sequence obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P6-1 vector with the pegRNA-06 target sequence and the RT&PBS sequence on pegRNA-06, respectively, while keeping other sequences unchanged.

[0161] The nucleotide sequence of the PE-P6-RTM-OsACC-1 recombinant expression vector (denoted as PE-P6-7 vector) is the sequence obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P6-1 vector with the pegRNA-07 target sequence and the RT&PBS sequence on pegRNA-07, respectively, while keeping other sequences unchanged.

[0162] The nucleotide sequence of the PE-P6-RTM-OsGS3 recombinant expression vector (denoted as PE-P6-8 vector) is the sequence obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P6-1 vector with the pegRNA-08 target sequence and the RT&PBS sequence on pegRNA-08, respectively, while keeping other sequences unchanged.

[0163] The nucleotide sequence of the PE-P7-RTM-OsALS-3 recombinant expression vector (denoted as PE-P7-1 vector) is obtained by inserting the 8-bp linker and mpknot motif (sequence 9) of the pegRNA-01 target between the RT&PBS sequence and the HDV sequence of the PE-P5-1 vector, while keeping the other sequences unchanged.

[0164] The nucleotide sequence of the PE-P7-RTM-OsEPSPS-2 recombinant expression vector (referred to as the PE-P7-2 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-02 target sequence and the RT&PBS sequence on the pegRNA-02, respectively, while keeping other sequences unchanged.

[0165] The nucleotide sequence of the PE-P7-RTM-OsGRF4-T2 recombinant expression vector (referred to as PE-P7-3 vector) is obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P7-1 vector with the pegRNA-03 target sequence and the RT&PBS sequence on pegRNA-03, respectively, while keeping other sequences unchanged.

[0166] The nucleotide sequence of the PE-P7-RTM-OsSD1 recombinant expression vector (referred to as PE-P7-4 vector) is obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P7-1 vector with the pegRNA-04 target sequence and the RT&PBS sequence on pegRNA-04, respectively, while keeping other sequences unchanged.

[0167] The nucleotide sequence of the PE-P7-RTM-OsCold1 recombinant expression vector (referred to as PE-P7-5 vector) is obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P7-1 vector with the pegRNA-05 target sequence and the RT&PBS sequence on pegRNA-05, respectively, while keeping other sequences unchanged.

[0168] The nucleotide sequence of the PE-P7-RTM-OsALS-1 recombinant expression vector (referred to as PE-P7-6 vector) is the sequence obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P7-1 vector with the pegRNA-06 target sequence and the RT&PBS sequence on pegRNA-06, respectively, while keeping other sequences unchanged.

[0169] The nucleotide sequence of the PE-P7-RTM-OsACC-1 recombinant expression vector (denoted as PE-P7-7 vector) is the sequence obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P7-1 vector with the pegRNA-07 target sequence and the RT&PBS sequence on pegRNA-07, respectively, while keeping other sequences unchanged.

[0170] The nucleotide sequence of the PE-P7-RTM-OsGS3 recombinant expression vector (denoted as PE-P7-8 vector) is obtained by replacing the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 in the PE-P7-1 vector with the pegRNA-08 target sequence and the RT&PBS sequence on pegRNA-08, respectively, while keeping other sequences unchanged.

[0171] PE-P6 OE hMLHdn RT-S-OsEPSPS-2 recombinant expression vector (denoted as PE-P6 OE hMLHdn The nucleotide sequence of the RT-S-1 vector) is an expression cassette consisting of an OsAct1-01 promoter sequence (sequence 10), a hMLH1dn gene sequence (sequence 11) and a Nos terminator sequence in sequence inserted between the Nos terminator sequence and the E35s promoter sequence of the PE-P5-1 vector, and the pegRNA-01 target sequence and the RT&PBS sequence on pegRNA-01 are replaced with the pegRNA-13 target sequence and the RT&PBS sequence on pegRNA-13, respectively, and the 8-bp linker and tevopreQ1 motif (sequence 9) of the pegRNA-13 target are inserted between the RT&PBS sequence and the HDV sequence, and the other sequences are kept unchanged.

[0172] PE-P6 OE hMLHdn RT-S-OsGRF4-T2 recombinant expression vector (denoted as PE-P6 OE hMLHdn RT-S-2) is PE-P6 OE hMLHdnThe sequence obtained by replacing the pegRNA-13 target sequence and the RT&PBS sequence on pegRNA-13 in the RT-S-1 vector with the pegRNA-14 target sequence and the RT&PBS sequence on pegRNA-14, respectively, while keeping other sequences unchanged.

[0173] PE-P6 OE hMLHdn RT-S-OsSD1 recombinant expression vector (denoted as PE-P6 OE hMLHdn RT-S-3) is PE-P6 OE hMLHdn The sequence obtained by replacing the pegRNA-12 target sequence and the RT&PBS sequence on pegRNA-12 in the RT-S-1 vector with the pegRNA-15 target sequence and the RT&PBS sequence on pegRNA-15, respectively, while keeping other sequences unchanged.

[0174] PE-P6 OE hMLHdn RT-M-OsEPSPS-2 recombinant expression vector (denoted as PE-P6- OE hMLHdn The nucleotide sequence of the RT-M-1 vector) is an expression cassette consisting of the OsAct1-01 promoter sequence (sequence 10), the hMLH1dn gene sequence (sequence 11) and the Nos terminator sequence in sequence inserted between the Nos terminator sequence and the E35s promoter sequence of the PE-P5-1 vector, and the pegRNA-01 target sequence and the RT&PBS sequence on the pegRNA-01 are replaced with the pegRNA-02 target sequence and the RT&PBS sequence on the pegRNA-02, respectively, and the 8-bplinker and tevopreQ1 motif (sequence 9) of the pegRNA-13 target are inserted between the RT&PBS sequence and the HDV sequence, and the other sequences are kept unchanged.

[0175] PE-P6 OE hMLHdn RT-M-OsGRF4-T2 recombinant expression vector (denoted as PE-P6 OE hMLHdn RT-M-2 vector) is used to transfer PE-P6 OE hMLHdn The sequence obtained by replacing the pegRNA-02 target sequence and the RT&PBS sequence on pegRNA-02 in the RT-M-1 vector with the pegRNA-03 target sequence and the RT&PBS sequence on pegRNA-03, respectively, while keeping other sequences unchanged.

[0176] PE-P6OE hMLHdn RT-M-OsSD1 recombinant expression vector (denoted as PE-P6 OE hMLHdn RT-M-3 vector) is used to transfer PE-P6 OE hMLHdn The sequence obtained by replacing the pegRNA-02 target sequence and the RT&PBS sequence on pegRNA-02 in the RT-M-1 vector with the pegRNA-04 target sequence and the RT&PBS sequence on pegRNA-04, respectively, while keeping other sequences unchanged.

[0177] PE-P6 ΔOsMLH1 RT-S-OsEPSPS-2 recombinant expression vector (denoted as PE-P6 ΔOsMLH1 The nucleotide sequence of RT-S-1 vector is PE-P6 OE hMLHdn The expression cassette composed of the OsAct1-01 promoter sequence (sequence 10), the gene sequence of hMLH1dn (sequence 11) and the Nos terminator sequence in the RT-S-1 vector was replaced with a DNA fragment composed of the OsU6a promoter and the DNA molecule shown in sequence 12 (tRNA+MLH1-T1 target sequence+esgRNA backbone sequence+tRNA+MLH1-T2 target sequence+esgRNA backbone sequence+PolyT), and the sequence obtained by keeping other sequences unchanged. In sequence 12, positions 1-77 are tRNA, positions 78-97 are MLH1-T1 target sequence, positions 98-183 are esgRNA backbone sequence, positions 184-260 are tRNA, positions 261-280 are MLH1-T2 target sequence, positions 281-366 are esgRNA backbone sequence, and positions 367-372 are PolyT.

[0178] PE-P6 ΔOsMLH1 RT-S-OsGRF4-T2 recombinant expression vector (denoted as PE-P6 ΔOsMLH1 RT-S-2 vector) is used to transfer PE-P6 ΔOsMLH1 The sequence obtained by replacing the pegRNA-13 target sequence and the RT&PBS sequence on pegRNA-13 in the RT-S-1 vector with the pegRNA-14 target sequence and the RT&PBS sequence on pegRNA-14, respectively, while keeping other sequences unchanged.

[0179] PE-P6 ΔOsMLH1 RT-S-OsSD1 recombinant expression vector (denoted as PE-P6 ΔOsMLH1 RT-S-3 vector) is used to transfer PE-P6 ΔOsMLH1The pegRNA-13 target sequence and the RT&PBS sequence on pegRNA-13 in the RT-S-1 vector are replaced with the pegRNA-15 target sequence and the RT&PBS sequence on pegRNA-15, respectively, while keeping other sequences unchanged.

[0180] PE-P6 ΔOsMLH1 RT-M-OsEPSPS-2 recombinant expression vector (denoted as PE-P6 ΔOsMLH1 The nucleotide sequence of RT-M-1 vector is PE-P6 ΔOsMLH1 The sequence obtained by replacing the pegRNA-13 target sequence and the RT&PBS sequence on pegRNA-13 in the RT-S-1 vector with the pegRNA-02 target sequence and the RT&PBS sequence on pegRNA-02, respectively, while keeping other sequences unchanged.

[0181] PE-P6 ΔOsMLH1 RT-M-OsGRF4-T2 recombinant expression vector (denoted as PE-P6 ΔOsMLH1 RT-M-2 vector) is used to transfer PE-P6 ΔOsMLH1 The sequence obtained by replacing the pegRNA-02 target sequence and the RT&PBS sequence on pegRNA-02 in the RT-M-1 vector with the pegRNA-03 target sequence and the RT&PBS sequence on pegRNA-03, respectively, while keeping other sequences unchanged.

[0182] PE-P6 ΔOsMLH1 RT-M-OsSD1 recombinant expression vector (denoted as PE-P6 ΔOsMLH1 RT-M-3 vector) is used to transfer PE-P6 ΔOsMLH1 The sequence obtained by replacing the pegRNA-02 target sequence and the RT&PBS sequence on pegRNA-02 in the RT-M-1 vector with the pegRNA-04 target sequence and the RT&PBS sequence on pegRNA-04, respectively, while keeping other sequences unchanged.

[0183] The esgRNA target sequence and RT&PBS sequence on the pegRNA in the above-mentioned vectors, as well as the 8-bp linker sequence are shown in Table 1.

[0184] Table 1

[0185]

[0186] 2. Obtaining Rice Resistance Callus and Positive T0 Seedlings

[0187] Step 1 was used to construct eight expression vectors of guide editing systems PE-P3, PE-P4, PE-P5, PE-P6, and PE-P7, as well as eight expression vectors of guide editing system PE-P6. ΔOsMLH1 RT-S and PE-P6 ΔOsMLH1 There are 3 expression vectors in each RT-M, a total of 46 recombinant expression vectors, and the operations are performed according to the following steps 1-9:

[0188] 1. Introduce the vector into Agrobacterium EHA105 (product of Shanghai Weidi Biotechnology Co., Ltd., CAT#: AC1010) to obtain recombinant Agrobacterium.

[0189] 2. Use medium (YEP medium containing 50 μg / mL kanamycin and 25 μg / mL rifampicin) to culture recombinant Agrobacterium at 28°C and 150 rpm shaking until OD 600 The concentration of glucose and sucrose in the infection medium was 10 g / L and 20 g / L, respectively. 600 is 0.2, and the Agrobacterium infection solution is obtained.

[0190] 3. Mature seeds of rice variety Nipponbare were shelled and threshed, placed in a 100 mL conical flask, added with 70% (v / v) ethanol aqueous solution and soaked for 30 seconds, then placed in a 25% (v / v) sodium hypochlorite aqueous solution, shaken and sterilized at 120 rpm for 30 minutes, rinsed with sterile water for 3 times, and dried with filter paper. Then, the seeds were placed with the embryo facing downward on N6 solid culture medium, and cultured in the dark at 28°C for 4-6 weeks to obtain rice callus.

[0191] 4. After completing step 3, soak the rice callus in Agrobacterium infection solution A (Agrobacterium infection solution A is a liquid obtained by adding acetosyringone to the Agrobacterium infection solution, and the amount of acetosyringone added satisfies the volume ratio of acetosyringone to the Agrobacterium infection solution of 25 μL:50 mL) for 10 minutes, then place it on a culture dish (containing about 200 ml of infection solution without Agrobacterium) covered with two layers of sterilized filter paper, and culture it in the dark at 21°C for 1 day.

[0192] 5. Place the rice callus obtained in step 4 on a recovery medium and culture it in the dark at 25-28°C for 3 days.

[0193] 6. Take the rice callus obtained in step 5, place it on the screening medium, and culture it in the dark at 28°C for 2 weeks.

[0194] 7. Take the rice callus obtained in step 6, place it on the screening medium again, and culture it in the dark at 28° C. for 2 weeks to obtain rice resistant callus.

[0195] 8. Take the rice resistant callus obtained in step 7 and place it on a differentiation medium, and culture it under light at 25°C for about 1 month. Move the differentiated seedlings to a rooting medium and culture them under light at 25°C for 2 weeks to obtain rice T0 seedlings.

[0196] 9. For the recombinant expression vectors 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-OsGS34, the genomic DNA of the obtained rice T0 seedlings was extracted and used as a template. The primers F (5'-TAC The method comprises the following steps: performing PCR amplification using a primer pair consisting of primer A (5'-GATGTTGGCGACCTCGTAT-3') and primer R (5'-GATGTTGGCGACCTCGTAT-3') to obtain a PCR amplification product; performing agarose gel electrophoresis on the PCR amplification product, and then making the following judgment: if the PCR amplification product contains a DNA fragment of about 973 bp, the corresponding rice T0 seedling is a rice-positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973 bp, the corresponding rice T0 seedling is not a rice-positive T0 seedling.

[0197] For the 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 recombinant expression vectors, genomic DNA of the obtained rice T0 seedlings was extracted and used as a template, and primers F (5'-TACTC The method comprises the following steps: performing PCR amplification using a primer pair consisting of primer A (5'-TCATCCACCAGTCCATC-3') and primer R (5'-GATGTTGGCGACCTCGTAT-3') to obtain a PCR amplification product; performing agarose gel electrophoresis on the PCR amplification product, and then making the following judgment: if the PCR amplification product contains a DNA fragment of about 973 bp, the corresponding rice T0 seedling is a rice-positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973 bp, the corresponding rice T0 seedling is not a rice-positive T0 seedling.

[0198] For the 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 recombinant expression vectors, genomic DNA of the obtained rice T0 seedlings was extracted and used as a template, and primers F (5'-TACTC The method comprises the following steps: performing PCR amplification using a primer pair consisting of primer A (5'-TCATCCACCAGTCCATC-3') and primer R (5'-GATGTTGGCGACCTCGTAT-3') to obtain a PCR amplification product; performing agarose gel electrophoresis on the PCR amplification product, and then making the following judgment: if the PCR amplification product contains a DNA fragment of about 973 bp, the corresponding rice T0 seedling is a rice-positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973 bp, the corresponding rice T0 seedling is not a rice-positive T0 seedling.

[0199] For 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-OsALS-1, PE-P5-RTM-OsACC-1, and PE-P5-RTM-OsGS3 recombinant expression vectors, genomic DNA of the obtained rice T0 seedlings was extracted and used as a template, and primers F (5'-TACTC The method comprises the following steps: performing PCR amplification using a primer pair consisting of primer A (5'-TCATCCACCAGTCCATC-3') and primer R (5'-GATGTTGGCGACCTCGTAT-3') to obtain a PCR amplification product; performing agarose gel electrophoresis on the PCR amplification product, and then making the following judgment: if the PCR amplification product contains a DNA fragment of about 973 bp, the corresponding rice T0 seedling is a rice-positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973 bp, the corresponding rice T0 seedling is not a rice-positive T0 seedling.

[0200] For the 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 recombinant expression vectors, genomic DNA of the obtained rice T0 seedlings was extracted and used as a template, and primers F (5'-TACTC The method comprises the following steps: performing PCR amplification using a primer pair consisting of primer A (5'-TCATCCACCAGTCCATC-3') and primer R (5'-GATGTTGGCGACCTCGTAT-3') to obtain a PCR amplification product; performing agarose gel electrophoresis on the PCR amplification product, and then making the following judgment: if the PCR amplification product contains a DNA fragment of about 973 bp, the corresponding rice T0 seedling is a rice-positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973 bp, the corresponding rice T0 seedling is not a rice-positive T0 seedling.

[0201] For 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, PE-P7-RTM-OsACC-1, and PE-P7-RTM-OsGS3 recombinant expression vectors, genomic DNA of the obtained rice T0 seedlings was extracted and used as a template, and primers F (5'-TACTC The method comprises the following steps: performing PCR amplification using a primer pair consisting of primer A (5'-TCATCCACCAGTCCATC-3') and primer R (5'-GATGTTGGCGACCTCGTAT-3') to obtain a PCR amplification product; performing agarose gel electrophoresis on the PCR amplification product, and then making the following judgment: if the PCR amplification product contains a DNA fragment of about 973 bp, the corresponding rice T0 seedling is a rice-positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973 bp, the corresponding rice T0 seedling is not a rice-positive T0 seedling.

[0202] For PE-P6 OE hMLHdn RT-S-OsEPSPS-2, PE-P6 OE hMLHdn RT-S-OsGRF4-T2, PE-P6 OE hMLHdnThe RT-S-OsSD1 recombinant expression vector is used, the genomic DNA of the obtained rice T0 seedlings is extracted and used as a template, and a primer pair consisting of primer F (5'-TACTCTCATCCACCAGTCCATC-3') and primer R (5'-GATGTTGGCGACCTCGTAT-3') is used for PCR amplification to obtain a PCR amplification product; the PCR amplification product is subjected to agarose gel electrophoresis, and then the following judgment is made: if the PCR amplification product contains a DNA fragment of about 973 bp, the corresponding rice T0 seedling is a rice-positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973 bp, the corresponding rice T0 seedling is not a rice-positive T0 seedling.

[0203] For PE-P6 OE hMLHdn RT-M-OsEPSPS-2, PE-P6 OE hMLHdn RT-M-OsGRF4-T2, PE-P6 OE hMLHdn The RT-M-OsSD1 recombinant expression vector is used, the genomic DNA of the obtained rice T0 seedlings is extracted and used as a template, and a primer pair consisting of primer F (5'-TACTCTCATCCACCAGTCCATC-3') and primer R (5'-GATGTTGGCGACCTCGTAT-3') is used for PCR amplification to obtain a PCR amplification product; the PCR amplification product is subjected to agarose gel electrophoresis, and then the following judgment is made: if the PCR amplification product contains a DNA fragment of about 973 bp, the corresponding rice T0 seedling is a rice-positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973 bp, the corresponding rice T0 seedling is not a rice-positive T0 seedling.

[0204] For PE-P6 ΔOsMLH1 RT-S-OsEPSPS-2, PE-P6 ΔOsMLH1 RT-S-OsGRF4-T2, PE-P6 ΔOsMLH1The RT-S-OsSD1 recombinant expression vector is used, the genomic DNA of the obtained rice T0 seedlings is extracted and used as a template, and a primer pair consisting of primer F (5'-TACTCTCATCCACCAGTCCATC-3') and primer R (5'-GATGTTGGCGACCTCGTAT-3') is used for PCR amplification to obtain a PCR amplification product; the PCR amplification product is subjected to agarose gel electrophoresis, and then the following judgment is made: if the PCR amplification product contains a DNA fragment of about 973 bp, the corresponding rice T0 seedling is a rice-positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973 bp, the corresponding rice T0 seedling is not a rice-positive T0 seedling.

[0205] 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, the genomic DNA of the obtained rice T0 seedlings is extracted and used as a template, and a primer pair consisting of primer F (5'-TACTCTCATCCACCAGTCCATC-3') and primer R (5'-GATGTTGGCGACCTCGTAT-3') is used for PCR amplification to obtain a PCR amplification product; the PCR amplification product is subjected to agarose gel electrophoresis, and then the following judgment is made: if the PCR amplification product contains a DNA fragment of about 973 bp, the corresponding rice T0 seedling is a rice-positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973 bp, the corresponding rice T0 seedling is not a rice-positive T0 seedling.

[0206] 3. Analysis of editing efficiency in rice callus and T0 seedlings

[0207] 1. In PE-P3 to PE-P7, 32 resistant calli obtained in step 7 of step 2 were randomly selected for each vector. After DNA was extracted, DNA of 8 calli were randomly mixed to finally obtain 4 mixed DNAs, which were divided into 4 groups. Using the mixed DNA as a template, for the OsALS-3 target site, primer F (5'-CTCCAGGGCCATACTTGTTG-3') and primer R (5'-TGGGTCATTCAGGTCAAACA-3') were used to perform PCR amplification on the sequence near the OsALS-3 target site to obtain the first round of PCR amplification products; for the OsEPSPS-2 target site, primer F (5'-GGCTCTCTGTGGAAGCAGAT-3') and primer R (5'-CATACGTTGCATTTCCACCA-3') were used to perform PCR amplification on the sequence near the OsEPSPS-2 target site. For the OsGRF4-T2 target site, primers F (5'-CCATTTTCTTGGCTCCAGTG-3') and R (5'-CTGCTCCAGCTCCTCGTACT-3') were used to amplify the sequence near the OsGRF4-T2 target site to obtain the first round of PCR amplification products; for the OsSD1 target site, primers F (5'-CGTGTCCGGCTACACCAG-3') and R (5'-AATGTCGTCCACCATCGTTT-3') were used to amplify the sequence near the OsSD1 target site to obtain the first round of PCR amplification products. For the OsCold1 target site, primers F (5'-CTATGCTATGCGTGCCAATC-3') and R (5'-CCTCTCCATCTCCATTTTGG-3') were used to amplify the sequence near the OsCold1 target site to obtain the first round of PCR amplification products; for the OsALS-1 target site, primers F (5'-TTGAGAACCTCCCTGTGAAG-3') and R (5'-TGAACCCCTTAGCAATAGTCACA-3') were used to amplify the sequence near the OsALS-1 target site to obtain the first round of PCR amplification products. PCR amplification was performed to obtain the first round of PCR amplification products; for the OsACC-1 target site, primer F (5'-TCTACATTCCCATGGCTGC-3') and primer R (5'-CCAGAACATTGCCTTTTGC-3') were used for PCR amplification to obtain the first round of PCR amplification products; for the OsGS3 target site, primer F (5'-AATCATGGATTTTGGCTTGG-3') and primer R (5'-GCTTCTCCGATGAACTGCTT-3') were used to PCR amplify the sequence near the OsGS3 target site to obtain the first round of PCR amplification products.Using the first round of PCR products as templates, different forward and reverse barcodes were added to the ends of the PCR products for library construction to form mixed libraries, which were sequenced using the MiSeq high-throughput sequencing platform, with a sequencing depth of more than 5000X for each mixed library (Qingke Biotechnology Co., Ltd.). The sequencing results were analyzed for each pegRNA region using CRISPResso2 (https: / / doi.org / 10.1038 / s41587-019-0032-3), and the editing efficiency of the guide editing system in rice callus was calculated. The editing efficiency of the guide editing system in rice callus is the average of the four groups of the proportion of the number of reads with all mutation sites detected to the total number of reads.

[0208] Results Figure 3 The results showed that in PE-P3 to PE-P7, the editing efficiencies of the OsALS-3 target in rice callus were 23.7%, 15.85%, 10.375%, 17.775% and 0, respectively; the editing efficiencies of the OsEPSPS-2 target in rice callus were 6.75%, 7.4%, 12.675%, 32.525%, and 0.925%, respectively; the editing efficiencies of the OsGRF4-T2 target in rice callus were 0.5%, 0, 2.9%, 5.425% and 0, respectively; the editing efficiencies of the OsSD1 target in rice callus were 4.275%, 1%, 27 .7%, 27.175% and 0; the editing efficiency of OsCold1 target in rice callus was 0, 0, 0, 19.2% and 0 respectively; the editing efficiency of OsALS-1 target in rice callus was 26.375%, 5.1%, 0.35%, 27.425% and 0.575% respectively; the editing efficiency of OsACC-1 target in rice callus was 0.45%, 0.275%, 3.95%, 36.975% and 1.925% respectively; the editing efficiency of OsGS3 target in rice callus was 2.6%, 2.5%, 0, 32.25% and 1.625% respectively. In summary, the guide editing system PE-P6 greatly improves the editing efficiency of rice targets in callus.

[0209] 2. In PE-P3 to PE-P7, the genomic DNA of the rice positive T0 seedling obtained in step 9 of step 1 was used as a template for each vector. For the OsALS-3 target site, the primer pair OsALS-3 was used for PCR amplification to obtain the first round of PCR amplification products; for the OsEPSPS-2 target site, the primer pair OsEPSPS-2 was used for PCR amplification to obtain the first round of PCR amplification products; for the OsGRF4-T2 target site, the primer pair OsGRF4-T2 was used for PCR amplification to obtain the first round of PCR amplification products; for the OsSD1 target site, the primer pair The primers for OsSD1 were PCR amplified to obtain the first round of PCR amplification products; for the OsCold1 target site, the primers for OsCold1 were PCR amplified to obtain the first round of PCR amplification products; for the OsALS-1 target site, the primers for OsALS-1 were PCR amplified to obtain the first round of PCR amplification products; for the OsACC-1 target site, the primers for OsACC-1 were PCR amplified to obtain the first round of PCR amplification products; for the OsGS3 target site, the primers for OsGS3 were PCR amplified to obtain the first round of PCR amplification products. Using the first round of PCR products as templates, different forward and reverse barcodes were added to the ends of the PCR products for library construction to form a mixed library, which was sequenced using the MiSeq high-throughput sequencing platform, and the sequencing depth of each mixed library was more than 1000X (Qingke Biotechnology Co., Ltd.). Each pegRNA region was analyzed, and the editing efficiency of the guide editing system in rice T0 seedlings was calculated. The editing efficiency of the guide editing system in rice T0 seedlings = the number of positive T0 seedlings with mutations at all mutation sites / the total number of positive T0 seedlings analyzed × 100%.

[0210] The results are shown in Table 2. The results showed that in PE-P3 to PE-P7, the editing efficiencies of the OsALS-3 target in rice T0 seedlings were 68.0%, 60%, 39.3%, 73% and 0, respectively; the editing efficiencies of the OsEPSPS-2 target in rice T0 seedlings were 79.7%, 54.0%, 70.4%, 91.5%, and 0, respectively; the editing efficiencies of the OsGRF4-T2 target in rice T0 seedlings were 20.6%, 1.7%, 19.6%, 54.5% and 11.3%, respectively; the editing efficiencies of the OsSD1 target in rice T0 seedlings were 2.2%, 2.9%, 37. The editing efficiencies of OsCold1 target in rice T0 seedlings were 0, 0, 1.4%, 60.2% and 2.7%, respectively; the editing efficiencies of OsALS-1 target in rice T0 seedlings were 76.0%, 33.7%, 2.4%, 50.6% and 0, respectively; the editing efficiencies of OsACC-1 target in rice T0 seedlings were 4.2%, 14.9%, 18.8%, 58.3% and 37.5%, respectively; the editing efficiencies of OsGS3 target in rice T0 seedlings were 3.5%, 18.6%, 5.3%, 56.3% and 3.3%, respectively. In summary, compared with PE-P3, PE-P4, PE-P5 and PE-P7, the editing efficiency of the eight rice targets in rice T0 seedlings was the highest after being edited by the guide editing system PE-P6.

[0211] 3. In PE-P6 OE hMLHdn RT-S, PE-P6 OE hMLHdn RT-M, PE-P6 ΔOsMLH1 RT-S and PE-P6 ΔOsMLH1In RT-M, the genomic DNA of the rice positive T0 seedlings obtained in step 9 of step 1 was used as a template for each vector. For the OsEPSPS-2 target site, primer F (5'-GGCTCTCTGTGGAAGCAGAT-3') and primer R (5'-CATACGTTGCATTTCCACCA-3') were used to PCR amplify the OsEPSPS-2 target site attachment sequence to obtain the first round of PCR amplification products; for the OsGRF4-T2 target site, primer F (5'-CCATTTTCTTGGCTCCAGTG-3') and primer R (5'-CTGCTCCAGCTCCTCGTACT-3') were used to PCR amplify the sequence near the OsGRF4-T2 target site to obtain the first round of PCR amplification products; for the OsSD1 target site, primer F (5'-CGTGTCCGGCTACACCAG-3') and primer R (5'-AATGTCGTCCACCATCGTTT-3') were used to PCR amplify the sequence near the OsSD1 target site to obtain the first round of PCR amplification products. Using the first round of PCR products as templates, different forward and reverse barcodes were added to the ends of the PCR products for library construction to form a mixed library, which was sequenced using the MiSeq high-throughput sequencing platform, and the sequencing depth of each mixed library was more than 1000X (Qingke Biotechnology Co., Ltd.). Each pegRNA region was analyzed, and the editing efficiency of the guide editing system in rice T0 seedlings was calculated. The editing efficiency of the guide editing system in rice T0 seedlings = the number of positive T0 seedlings with mutations in all mutation sites / the total number of positive T0 seedlings analyzed × 100%.

[0212] The results are shown in Table 3. The results show that in PE-P6, 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 efficiencies of OsEPSPS-2 target in rice T0 seedlings were 84.3%, 60.5.8%, 77.1%, 93.5% and 92.5%, respectively; the editing efficiencies of OsGRF4-T2 target in rice T0 seedlings were 56.8%, 40.0%, 64.6%, 66.6% and 85.5%, respectively; the editing efficiencies of OsSD1 target in rice T0 seedlings were 44.8%, 64.0%, 73.8%, 64.0% and 86.5%, respectively. In summary, compared with the guide editing system PE-P6, the guide editing efficiency was not improved after overexpression of the hMLHdn gene; while after knocking out the OsMLH1 gene, the guide editing system PE-P6 ΔOsMLH1 RT-S and PE-P6 ΔOsMLH1RT-M can further improve the editing efficiency of the target; and guide the editing system PE-P6 ΔOsMLH1 RT-M can maximize the editing efficiency of rice targets.

[0213] Table 2

[0214]

[0215] Table 3

[0216]

[0217] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A complete system, comprising a fusion protein, esgRNA and pegRNA; The fusion protein includes reverse transcriptase, Cas9 nicking enzyme, self-cleaving oligopeptide and screening marker protein in sequence; The Cas9 nicking enzyme is Cas9maxn; the Cas9maxn is a protein whose amino acid sequence is shown in Sequence 5; The reverse transcriptase is M-MLVRT; the M-MLVRT is a protein whose amino acid sequence is shown in Sequence 1; The self-cleaving oligopeptide is a 2A self-cleaving oligopeptide derived from a viral genome; the 2A self-cleaving oligopeptide derived from a viral genome is a 2A self-cleaving oligopeptide derived from porcine teschovirus-1; the 2A self-cleaving oligopeptide derived from porcine teschovirus-1 is a protein whose amino acid sequence is shown in Sequence 3; The esgRNA targets the target sequence of the MLH1 gene in rice; The pegRNA includes esgRNA', a reverse transcription template sequence, a primer binding site sequence, a connection sequence and a tevopreQ1 motif in sequence; the esgRNA' targets the target gene target sequence; The connection sequence is 8-bp linker; The nucleotide sequence of the tevopreQ1 motif is as described in Sequence 8; The pegRNA is driven to express by a composite promoter; the composite promoter is composed of E35S promoter, CmYLCV promoter and OsU3 promoter in sequence; The nucleotide sequence of the E35S promoter is shown in SEQ ID NO: 10025-10461; The nucleotide sequence of the CmYLCV promoter is shown in SEQ ID NO: 10462-10920; The nucleotide sequence of the OsU3 promoter is shown in SEQ ID NO: 10932-11270.

2. The complete system according to claim 1, characterized in that: The esgRNA consists of an MLH1 gene target sequence and an esgRNA skeleton; Alternatively, the esgRNA' consists of a target gene target sequence and an esgRNA backbone.

3. The complete system according to claim 1 or 2, characterized in that: The selection marker protein is hygromycin phosphotransferase; The hygromycin phosphotransferase is a protein whose amino acid sequence is shown in Sequence 4.

4. Application of the complete system according to any one of claims 1 to 3 in any one of the following S1) to S4): S1) Editing of genome sequences of organisms or biological cells; S2) preparing an edited product of an organism or an organism cell genome sequence; S3) improving the editing efficiency of the genome sequence of an organism or biological cell; S4) preparing a product for improving the editing efficiency of the genome sequence of an organism or biological cell; The organism is rice; and the biological cell is a rice cell.

5. The use according to claim 4, characterized in that: The editing of the genome sequence is base replacement of the genome sequence.

6. A method for editing a genome sequence, comprising the following steps: causing an organism or a biological cell to express the fusion protein described in claim 1, the esgRNA described in claim 1, and the pegRNA described in claim 1; the organism is rice; and the biological cell is a rice cell.

7. A method for improving the editing efficiency of the genome sequence of an organism or a biological cell, comprising the following steps: causing the organism or the biological cell to express the fusion protein described in claim 1, the esgRNA described in claim 1, and the pegRNA described in claim 1; the organism is rice; and the biological cell is a rice cell.

8. A method for preparing a biological mutant, comprising the following steps: editing the genome sequence of an organism or a biological cell according to the method described in claim 6 or 7 to obtain a biological mutant; the organism is rice; and the biological cell is a rice cell.

9. The method according to any one of claims 6 to 8, characterized in that: The editing of the genome sequence is base replacement of the genome sequence.

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