CN113846075A - MAD7-NLS fusion protein, nucleic acid construct for site-directed editing of plant genome and application thereof - Google Patents

Abstract

The invention provides a MAD7-NLS fusion protein, which has the following structure: B1-C-B2, B1-C or C-B2, wherein C is MAD7 protein; b1 and B2 are each independently nuclear localization signal sequences (NLS). The invention also provides a nucleic acid construct for site-directed editing of a plant genome, comprising a first expression cassette comprising, in sequence, a first promoter, a nucleotide sequence encoding a MAD7-NLS fusion protein, and a first terminator. The invention also provides application of the nucleic acid construct in plant gene editing. The invention can simply and efficiently carry out single gene knockout, multiple gene knockout or homologous recombination and targeted insertion of exogenous fragments at a preset plant genome site by utilizing the MAD 7.

Description

MAD7-NLS fusion protein, nucleic acid construct for site-directed editing of plant genome and application thereof

Technical Field

The invention relates to the field of biotechnology. In particular to a MAD7-NLS fusion protein, a nucleic acid construct for site-directed editing of plant genome and a high-efficiency site-directed gene editing method of plant genome guided by RNA based on a novel nuclease MAD 7.

Background

With the popularization and application of gene-oriented editing tools such as Zinc finger protein ribonuclease (ZFN), Transcription activator-like effector nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems, especially CRISPR systems have been developed very rapidly in recent years due to their high activity, good specificity, simple and convenient operation, and are successfully applied to a large number of species. The most widely used nucleases at present are type II Cas9 and Cpf1 (Cas 12 a). Among these, Cas9 recognizes the 3 'G-rich site, while Cpf1 (Cas 12 a) recognizes the 5' a-T rich site. Compared with Cas9, Cas12a protein has functions of DNA-cleaving enzyme and RNA-cleaving enzyme, not only can target-cleave DNA double strand, but also can process and cleave corresponding non-mature crRNA (pre-crRNA) into mature crRNA (Fonfara et al, 2016), and does not need tracrRNA to participate in; meanwhile, the Cas12a protein molecule is relatively smaller, has stronger specificity, and has stronger advantage on multi-target gene editing compared with Cas 9; in addition, the cohesive end formed by Cas12a splicing the genome is more favorable for the directional insertion of the foreign gene than the blunt end formed by Cas9 splicing the genome.

MAD7 belongs to the type II V-A Cpf1-like family, is discovered in the genus Eubacterium by the company Inscripta, is optimized and modified, and can be freely used by scientific research institutions and commercial research. The homology of the MAD7 and the AsCpf1 protein is highest, the homology is only 31%, the PAM recognition site is YTTN, the MAD7 system has higher editing activity in bacteria, yeast, zebrafish, mice and human cells, and the editing efficiency of the MAD7 in plants is researched after the rice codon is optimized in order to popularize the application range of the MAD7 system in the plants. Although the MAD7 system has been reported to be applied to rice recently, the mutation efficiency is 49-65.6%. In summary, in order to meet the needs of plant genetic engineering and enrich the tool boxes for plant gene editing, it is necessary to develop a more efficient gene editing system with strong advantages.

Disclosure of Invention

The invention aims to provide a set of high-efficiency site-directed editing system of CRISPR/MAD7 plant genome based on MAD7, which can simply and efficiently realize single gene knockout, multiple gene knockout and homologous recombination or site-directed knock-in of exogenous fragments in single-cotyledon and double-cotyledon plants.

The invention firstly provides a MAD7-NLS fusion protein, which has the following structure:

B1-C-B2, B1-C or C-B2;

wherein,

c is MAD7 protein;

b1 and B2 are each independent nuclear localization signal sequences (NLS).

In one embodiment according to the invention, the nuclear localization signal sequence (NLS) is selected from: SV40, KRP2 (Kipredicted protein gene NO. 2), MDM2, CDc25C, DPP9, MTA1, CBP80, AreA, M9, Rev, hTAP, MyRF, EBNA-6, TERT, or Tfam, or a combination of any two or more thereof.

In one embodiment according to the invention, the N-terminus of the MAD7-NLS fusion protein further comprises a signal peptide and/or a protein tag sequence.

Another aspect of the present invention provides a nucleic acid construct for site-directed editing of a plant genome, comprising a first expression cassette comprising, in sequential linkage, a first promoter, a nucleotide sequence encoding a MAD7-NLS fusion protein as described above, and a first terminator;

preferably, the first promoter is a Pol II type promoter, preferably Ubi, Actin, CmYLCV, UBQ, 35S, SPL, one or a combination of any two of the tissue specific promoters YAO, CDC45, rbcS and inducible promoter XEV.

In one embodiment according to the present invention, the nucleic acid construct further comprises a second expression cassette comprising a second promoter, a plurality of tandem repeats, linked in sequence;

the repetitive sequence is one or two of a mature Direct Repeat (DR) and an immature direct repeat; preferably, the second promoter is a Pol II type or Pol III type promoter; more preferably, the second promoter is selected from one, two or more of OsU3, OsU6a, OsU6b, OsU6c, Actin, 35S, Ubi, UBQ, SPL, CmYLCV, tissue specific promoter YAO, CDC45, rbcS or inducible promoter XEV.

In one embodiment according to the invention, the second expression cassette further comprises a second termination sequence linked to the end of the repeat sequence;

preferably, the second termination sequence is selected from polyT, NOS, polyA, or a combination thereof.

In one embodiment according to the invention, the repeat sequence further comprises a target site leader sequence sg; preferably, the sg sequence has a length of 17-35bp, preferably 19-28bp, more preferably 19-25 bp;

preferably, the number of repeats of the repeat sequence is 2 to 50, preferably 2 to 10, more preferably 3 to 15, and further preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

Particularly preferably, the nucleic acid construct is one or more selected from the group consisting of SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 or SEQ ID NO 21.

In one embodiment according to the invention, the nucleic acid construct is a vector comprising both a first expression cassette and a second expression cassette, or,

a vector combination consisting of a first vector comprising the first expression cassette and a second vector comprising the second expression cassette, respectively.

The invention also provides a kit for gene editing in plants, which is characterized by comprising the nucleic acid construct; preferably, a helper vector carrying the donor DNA expression cassette is also included.

Yet another aspect of the present invention provides a method for gene editing in a plant, comprising:

(i) introducing the nucleic acid construct and optionally the donor nucleic acid fragment into a plant cell, plant tissue or plant body, and performing gene editing in said plant cell, plant tissue or plant body;

(ii) screening and identifying plant cells, plant tissues or plant bodies in which said gene editing has occurred;

(iii) (iii) regenerating or culturing the plant cells, plant tissues or plant bodies identified in step (ii) as having undergone said gene editing;

preferably, the gene editing comprises one of gene knockout, site-directed insertion or gene replacement, or optionally a combination of two or three;

preferably, the gene editing is single-site or multi-site gene editing;

preferably, the introduction is achieved using any one method selected from the group consisting of an agrobacterium transformation method, a particle gun method, a microinjection method, an electric shock method, an ultrasonic method, or a polyethylene glycol (PEG) mediated method;

preferably, the plant is selected from any one of gramineae, leguminous plants, solanaceae or cruciferae plants;

more preferably, the plant is selected from any one of arabidopsis, wheat, barley, oat, maize, rice, sorghum, millet, soybean, peanut, tobacco or tomato.

The technical scheme of the invention has the following beneficial effects:

the CRISPR/MAD7 plant genome high-efficiency site-specific editing system based on MAD7 can simply and efficiently realize single gene knockout, multiple gene knockout and homologous recombination or exogenous fragment site-specific knock-in on a single-cotyledon plant, and compared with the traditional CRISPR/Cas9 and CRISPR/Cpf1 gene editing systems, the CRISPR/MAD7 system provided by the invention has higher specificity and is more flexible in selection of target sites.

Drawings

FIG. 1 is a schematic diagram showing the structural composition of the elements in the related sequence (SEQ ID NO. 2) of the rice crRNA expression cassette of MAD7 according to the present invention.

FIG. 2 is a schematic diagram showing the structural composition of the elements in the rice polygenic site editing sg-DR crRNA expression cassette's related sequence (SEQ ID NO.3) according to MAD7 of the present invention.

FIG. 3 is a schematic diagram showing the structural composition of the elements in the rice polygenic site editing tRNA-Sg-DR crRNA expression cassette's related sequence (SEQ ID NO.4) according to MAD7 of the present invention.

FIG. 4 is a schematic diagram showing the structural composition of the elements in the rice polygenic site editing miniOsU3/U6-DR-sg crRNA expression cassette related sequence (SEQ ID NO.5) according to MAD7 of the present invention.

FIG. 5 is a schematic diagram showing the structural composition of the elements in the rice polygenic site editing HH-DR-sg-HDV crRNA expression cassette-related sequence (SEQ ID NO.6) according to MAD7 of the present invention.

FIG. 6 is a schematic diagram showing the structural composition of the elements in the relevant sequence (SEQ ID NO. 21) of the maize crRNA expression cassette for MAD7 according to the present invention.

FIG. 7 is a schematic diagram of construction of rice single-site knockout CRISPR-MAD7 (ncNLS) expression vector.

FIG. 8 is a schematic diagram of construction of expression vectors under three placement modes ' ' ' designed by NLS when OsHD3A site is knocked out at single site in rice.

FIG. 9 is a schematic diagram of construction of a rice multi-site knockout CRISPR-MAD7 expression vector containing a DR-sg sequence string.

FIG. 10 is a schematic diagram of construction of a rice multi-site-knockout CRISPR-MAD7 expression vector with tRNA tandem DR-sg sequence.

FIG. 11 is a schematic diagram of construction of CRISPR-MAD7 expression vectors with the drive DR-sg sequence of miniOsU3/miniOsU6, respectively.

FIG. 12 is a schematic diagram of the construction of CRISPR-MAD7 expression vector driven by Pol III type promoter and with HH-HDV tandem DR-sg sequence.

FIG. 13 is a schematic diagram of the construction of CRISPR-MAD7 expression vector driven by Pol II type promoter and with HH-HDV tandem DR-sg sequence.

FIG. 14 is a schematic diagram of the construction of a maize single-site knockout CRISPR-MAD7 (ncNLS) expression vector.

Detailed Description

In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.

The inventor conducts extensive and intensive research to construct a high-universality and high-specificity CRISPR/MAD7 plant genome site-directed efficient editing system based on MAD7, a nucleic acid construct, a vector or a vector combination for site-directed editing of plant genome, and a plant genome site-directed editing method. Specifically, based on the method of the present invention, single gene knockout, multiple gene knockout or homologous recombination and targeted insertion of foreign fragments can be performed easily and efficiently at a predetermined plant genomic site. On the basis of this, the present invention has been completed.

In particular, the CRISPR/MAD7 editing system of the invention adopts mature crRNA, and the development of the CRISPR-MAD7 system based on the mature crRNA is more advantageous because the mature crRNA is shorter, is convenient for artificial synthesis and is easier to transform into cells. On the basis, a technical scheme that a nuclear localization signal sequence (NLS) is placed at the N end and the C end of the MAD7 protein is developed. Experimental results show that the technical scheme is adopted to successfully finish the gene editing of the rice, and the efficiency is as high as 95%.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications and other references mentioned herein are incorporated herein by reference.

As used herein, the terms "comprising," having, "or" including "include" comprising, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … ….

As used herein, the term "operably linked" or "operably linked" refers to the condition wherein certain portions of a linear DNA sequence are capable of modulating or controlling the activity of other portions of the same linear DNA sequence. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.

Nucleic acid constructs and methods for efficient site-directed editing of plant genomes based on MAD7

The invention provides a nucleic acid construct for efficient site-directed editing of a plant genome, said nucleic acid construct comprising a first expression cassette and optionally a second expression cassette;

wherein the first expression cassette is an expression cassette of MAD7-NLS fusion protein, wherein the MAD7-NLS fusion protein has the structure of formula I:

P1-A-B1-C-B2-E1 （I）

in the formula,

p1 is a first promoter;

a is nothing, a signal peptide, and/or a protein tag sequence;

b1 is a null or nuclear localization signal sequence NLS;

b2 is a null or nuclear localization signal sequence NLS;

with the proviso that at most one of B1 and B2 is null;

c is MAD7 protein;

e1 is a first terminator;

the second expression cassette is a crRNA expression cassette which contains a coding sequence corresponding to mature crRNA or non-mature pre-crRNA.

In the present invention, the above-mentioned elements can be prepared by a conventional method (e.g., PCR method, artificial total synthesis) and then ligated by a conventional method to form the nucleic acid construct of the present invention. If desired, an enzymatic cleavage reaction may optionally be carried out prior to the ligation reaction.

In addition, the nucleic acid constructs of the invention may be linear or circular. The nucleic acid construct of the present invention may be single-stranded or double-stranded. The nucleic acid constructs of the invention may be DNA, RNA, or DNA/RNA hybrids.

As used herein, nlls refers to the nuclear localization signal sequence NLS located at the 5 'end of the MAD7 protein coding sequence, cNLS refers to the nuclear localization signal sequence NLS located at the 3' end of the MAD7 protein coding sequence, and ncNLS refers to the nuclear localization signal sequence NLS linked to both the 5 'end and the 3' end of the MAD7 protein coding sequence.

As used herein, "exogenous gene" refers to an exogenous DNA molecule whose action is a stepwise action. The foreign gene that can be used in the present application is not particularly limited, and includes various foreign genes commonly used in the field of transgenic animals. Representative examples include (but are not limited to): beta-glucuronidase gene, red fluorescent protein gene, green fluorescent protein gene, lysozyme gene, salmon calcitonin gene, lactoferrin, serum albumin gene, etc.

As used herein, "selectable marker gene" refers to a gene used in a transgenic process to select a transgenic cell or a transgenic animal, and the selectable marker gene that can be used in the present application is not particularly limited, and includes various selectable marker genes commonly used in the transgenic field, representative examples including (but not limited to): hygromycin resistance gene (Hyg), kanamycin resistance gene (NPTII), neomycin, or puromycin resistance gene.

The term "expression cassette" as used herein refers to a polynucleotide sequence comprising the sequence components of the gene to be expressed and the elements required for expression. For example, in the present invention, the term "selectable marker expression cassette" refers to a polynucleotide sequence comprising a sequence encoding a selectable marker and a sequence module for expressing a desired element. Components required for expression include a promoter and polyadenylation signal sequence. In addition, the selectable marker expression cassette may or may not contain other sequences, including (but not limited to): enhancers, secretory signal peptide sequences, and the like.

As used herein, the term "plant promoter" refers to a nucleic acid sequence capable of initiating transcription of a nucleic acid in a plant cell. The plant promoter may be derived from plants, microorganisms (such as bacteria, viruses) or animals, or may be a promoter artificially synthesized or modified.

The term "plant terminator" as used herein refers to a terminator capable of stopping transcription in a plant cell. The plant transcription terminator can be derived from plants, microorganisms (such as bacteria and viruses) or animals, or is a synthetic or modified terminator. Representative examples include (but are not limited to): nos terminator.

As used herein, the term "MAD 7 protein" refers to a nuclease. Typical MAD7 proteins include (but are not limited to):

ErCas12a （Eubacterium rectale）

as used herein, the term "coding sequence for MAD7 protein" refers to a nucleotide sequence that encodes MAD7 protein having cleavage activity. In the case where the inserted polynucleotide sequence is transcribed and translated to produce a functional MAD7 protein, the skilled artisan will recognize that, because of the degeneracy of the codons, a large number of polynucleotide sequences may encode the same polypeptide. In addition, the skilled artisan will recognize that different species have certain preferences for codons, and that the codons of MAD7 protein may be optimized as required for expression in different species, and such variants are specifically covered by the term "coding sequence for MAD7 protein". In addition, the term specifically includes full-length, substantially identical sequences to the MAD7 gene sequence, as well as sequences encoding proteins that retain the function of the MAD7 protein.

In the present invention, said C corresponds to the full length or fusion protein of MAD7 protein and said second expression cassette has the structural crRNA expression cassette of formula II:

P2-(R-S)q-T （II）

in the formula,

p2 is a second promoter;

each R is independently a Direct Repeat (DR) sequence corresponding to a mature or an immature form

Each S is independently a null or target site leader sequence sg;

q is a positive integer not less than 1;

t is none or polyT or Nos or polyA sequence.

The invention also provides a vector or vector combination comprising a nucleic acid construct of the invention.

Preferably, the vector combination of the present invention further comprises a helper vector carrying the donor DNA expression cassette.

In the nucleic acid constructs and/or vectors of the invention, some elements are operably linked to each other, particularly to the corresponding elements in each expression cassette. For example, a promoter, when operably linked to a coding sequence, means that the promoter is capable of initiating transcription of the coding sequence.

The invention also provides a reagent combination and a kit containing the vector or the vector combination, which can be used for the plant gene editing method.

The invention also provides a method for carrying out gene editing on plants, which comprises the following steps:

(i) introducing (a) a vector or combination of vectors of the invention and (b) optionally a donor nucleic acid fragment into a plant cell, plant tissue or plant (plant) to produce gene editing in said plant cell, plant tissue or plant; and

(ii) optionally, the plant cell or plant in which the gene editing occurs is detected, screened or identified.

In the present invention, the gene editing includes gene knockout, site-specific insertion, gene replacement, or a combination thereof.

The plant gene editing method can be used for improving various plants, particularly for improving crops.

As used herein, the term "plant" includes whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, and plant cells and progeny of same. The type of plant that can be used in the method of the invention is not particularly limited and generally includes any higher plant type that can be subjected to transformation techniques, including monocotyledonous, dicotyledonous and gymnosperms.

The invention can be used in the field of plant genetic engineering, such as plant gene function research and crop genetic improvement.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the laboratory Manual (New York: Cold Spring Harbor laboratory Press, 1989), or according to the manufacturer's recommendations. The experimental materials referred to in the present invention are commercially available without specific reference.

Material

The coding sequence of the MAD7 protein is a coding sequence for carrying out codon optimization on rice, and the specific sequence is shown in SEQ ID NO. 1.

The related sequence (SEQ ID NO. 2) of the rice crRNA expression cassette of the second MAD7 sequence, the element structure in the expression cassette is shown in figure 1, the AvrII and AfeI enzyme cutting sites are marked by underlining before and after the sequence respectively, and the sequence is set for convenient cloning to a pCAMBIA expression vector; black shading marks the mature DR sequence corresponding to MAD 7; the square frame is an sg locus, when a gene knockout vector is constructed, the sequence artificially synthesizes a complementary double strand, and the PCR product of the OsU3 promoter is amplified to full length by an Overlapping PCR method; bold letters are transcription terminator sequences; the remainder is the sequence of OsU3 promoter.

The rice polygene site of the three MAD7 edits the related sequence (SEQ ID NO.3) of the sg-DR crRNA expression cassette, the element structure in the expression cassette is shown in figure 2, wherein, the underlined marks BsaI enzyme cutting site, which is set for convenient cloning to pCAMBIA expression vector; black shading marks the mature DR sequence corresponding to MAD 7; the sg sequences of the four targeting sites in boxes are OsDEP1-g6, OsBEL260-g1, OsRoc5-g1 and OsHD3A in 5 'to 3' order; bold letters are transcription terminator sequences; the remainder is the sequence of OsU3 promoter.

The rice polygene site of the four MAD7 edits the related sequence (SEQ ID NO.4) of the tRNA-sg-DR crRNA expression cassette, the element structure in the expression cassette is shown in figure 3, wherein, the front underline and the back underline respectively mark AvrII and BsaI enzyme cutting sites, which are arranged for convenient cloning to pCAMBIA expression vector; black shading marks the mature DR sequence corresponding to MAD 7; the sg sequences of the four targeting sites in boxes are OsDEP1-g6, OsBEL260-g1, OsRoc5-g1 and OsHD3A in 5 'to 3' order; tRNA sequences are double underlined; bold letters are transcription terminator sequences; the remainder is the sequence of OsU3 promoter.

The rice polygene site of the five MAD7 edits a related sequence (SEQ ID NO.5) of a miniOsU3/U6-DR-sg crRNA expression cassette, the element structure in the expression cassette is shown in figure 4, wherein, front underlines and back underlines respectively mark AvrII and BsaI enzyme cutting sites, and the enzyme cutting sites are arranged for being conveniently cloned to a pCAMBIA expression vector; black shading marks the mature DR sequence corresponding to MAD 7; the sg sequences of the four targeting sites in boxes are OsDEP1-g6, OsBEL260-g1, OsRoc5-g1 and OsHD3A in 5 'to 3' order; bold letters are transcription terminator sequences; the rest is the sequence of the miniOsU3/U6 promoter.

The rice polygene site of the six MAD7 edits a related sequence (SEQ ID NO.6) of an HH-DR-sg-HDV crRNA expression cassette, the element structure in the expression cassette is shown in figure 5, wherein, the underlined marks a BsaI enzyme cutting site which is arranged for being conveniently cloned to a pCAMBIA expression vector; black shading marks the mature DR sequence corresponding to MAD 7; the sg sequences of the four targeting sites in boxes are OsDEP1-g6, OsBEL260-g1, OsRoc5-g1 and OsHD3A in 5 'to 3' order; bold letters are transcription terminator sequences; double underlined and dotted sequences are hammerhead (hh), and Hepatitis Deltavirus (HDV) ribozyme sequences, respectively.

Wherein, 5 'in HH nucleic acid sequence'tttgacComplementary to the first 6 bases of DR.

A related sequence (SEQ ID NO. 21) of a maize crRNA expression cassette of a sequence twenty-one MAD7, wherein the element structure in the expression cassette is shown in figure 6, ApaI enzyme cutting sites are respectively marked by front underline and back underline, and the ApaI enzyme cutting sites are arranged for convenient cloning to a pCAMBIA expression vector; black shading marks the mature DR sequence corresponding to MAD 7; the square frame is an sg locus, when a gene knockout vector is constructed, the sequence artificially synthesizes a complementary double strand, and the PCR product of the ZmU3 promoter is amplified to full length by an Overlapping PCR method; bold letters are transcription terminator sequences; the remainder is the sequence of ZmU3 promoter.

Method

1) Construction of CRISPR-MAD7 expression vector for single-site knockout

And performing rice codon optimization on the MAD7, constructing two expression cassettes of MAD7 and crRNA, and cloning the expression cassettes to a pCAMBIA expression vector. In the case of rice, the crRNA expression cassette (SEQ ID NO: 2) has the following 4 elements from 5 'to 3': OsU6 or OsU3 promoter, the mature Direct Repeat (DR) corresponding to MAD7, sg sequence, transcription terminator sequence (TTTTTTT). The MAD7 expression cassette has the following elements from 5 '-3': the Ubi promoter from maize, NLS nuclear localization signal sequence (nlls), coding sequence of MAD7, second NLS nuclear localization signal sequence (cNLS), NOS transcription terminator sequence (fig. 7). Another design of the present invention is to change the sg length, or to remove the NLS nuclear localization signal sequence at the 5 'or 3' end of the coding sequence of MAD7 based on the above construction, and to retain other elements (FIG. 8) to investigate the in vivo cleavage activity of MAD7 in different combinations.

2) Construction of CRISPR-MAD7 vector for multiple site knockout

Since MAD7 has RNase activity, it can self-cleave the transcribed precursor crRNA sequence, supposing that if a crRNA expression cassette is concatenated with multiple DR-sg sequences, it can be cleaved into individual DR-sg sequences by MAD7 after transcription, thereby conveniently realizing multi-site knock-out. The design needs to construct a multi-site crRNA expression cassette, taking rice as an example, and the multi-site crRNA expression cassette has the following elements from 5 'to 3': OsU6 or OsU3 promoter, a crRNA sequence (containing a DR sequence) corresponding to MAD7, an sg1 sequence corresponding to target site 1, a crRNA sequence, an sg2 sequence corresponding to target site 2, a crRNA sequence, an sg3 sequence corresponding to target site 3, a crRNA sequence, an sgN sequence corresponding to target site N, a transcription terminator sequence (TTTTTTT). Another design of the invention is that multiple DR-sg are driven by miniOsU3/U6 promoter respectively, or different DR-sg sequences are connected with tRNA processing recognition sequence or HH-HDV interval, and MAD7 expression cassette is constructed as single site knockout. Finally, the two expression cassettes of crRNA and MAD7 nuclease were cloned into pCAMBIA expression vector (FIGS. 9-13).

3) Homologous recombination or non-homologous end joining (NHEJ) -mediated site-directed insertion of foreign fragments using CRISPR-MAD 7.

The cleavage of MAD7 with Cpf1 produces sticky ends, theoretically more suitable for targeted insertion of foreign fragments than Cas9 cleavage produces blunt ends. The method is characterized in that a DSB is manufactured near a target site by using CRISPR-MAD7, and a large number of exogenous fragments are introduced by using a gene gun bombardment or DNA virus replication mode, so that the gene homologous recombination or directional insertion of plant cells can be efficiently realized.

Example 1 Single site knockout of endogenous Rice Gene Using CRISPR-MAD7 (ncNLS) System

The LbCpf1 is adopted to efficiently edit the OsHD3A promoter targeting site (LOCOs06g06320, editing efficiency 81.5%), and the promoter targeting site is respectively connected to the pCAMBIA-CRISPR-MAD7 expression vector through AvrII and Afel enzyme cutting sites. The constructed vector is transformed into agrobacterium EHA105, then the strain infects the callus of a rice variety Nanjing 46 (Oryza sativa ssp japonica cv. Nanging 46), the callus is recovered for 4 days at 30 ℃ and 34 ℃ respectively after 3 days of co-culture, the transformed callus is transferred to a screening culture medium containing hygromycin, the transformed callus is transferred to a differentiation culture medium containing hygromycin to regenerate a plant after 28 to 30 days of culture, the regenerated plant is sampled to extract DNA, Taqman detects MAD7 positive single plant, and performs amplification sequencing on a positive single plant target site. The results show that the system can generate mutation by targeted cutting in rice cells and generate mutant plants, the editing efficiency of the OsHD3A locus is 89.9 percent and 94.9 percent respectively when the rice cells are restored to be cultured at 30 ℃ and 34 ℃, the frequency (containing homo) of the simultaneous editing of two alleles is up to 77.5 percent and 78.1 percent, the editing efficiency is equivalent to that of Lcpcpf 1, and no significant difference exists between the editing efficiencies under two different temperature treatments (Table 1).

Table 1 PAM-sg sequence of single-site knockout of rice endogenous gene in CRISPR/MAD7 (ncNLS) system and identification result of T0 generation plant

Note: the number in brackets is the number of single plants without sequencing results; the PAM sequence is underlined; WT stands for wild type, He for heterozygous mutant, Bi for allelic double mutant (comprising Homo homozygous mutant). Editing efficiency (%) = number of mutants/(MAD 7)⁺Positive individual-number of failed sequenced strains) 100; biallelic frequency (%) = number of Biallelic strains/(MAD 7)⁺Positive individual-number of failed sequenced strains) 100.

Example 2 Single site knockout of multiple endogenous genes of Rice Using the CRISPR-MAD7 (ncNLS) System

To further verify the efficiency of targeted cleavage of CRISPR-MAD7 (ncNLS) system in plant cells, 5 article reports of editing sites of Cpf1 in rice (OsRLK-799-g 1, LOC _ Os02g07960; OsDEP1-g6, LOC _ Os09g26999; OsALS-g7, LOC _ Os02g30630; OsBEL260-g1, LOC _ Os03g55260; OsRoc5-g1, LOC _ Os02g 45250) were selected, and CRISPR-MAD7 targeting sites (OsDEP 1-g7; OsPDS1-g 3; LOC _ Os03g08570; OsRoc5-g2 and OsBEL260-g 4) were newly designed according to known gene sequences, artificially synthesized CRISPR-MAD-sgROV, and the product of the CRISPR-MAD-7-PCR amplification site was ligated to the plant cell amplification vector. The constructed vector is transformed into agrobacterium EHA105, then the callus of the rice variety Nanjing 46 is infected by the strain, the culture is recovered for 4 days at 30 ℃ after 3 days of culture, the transformed callus is transferred to a screening culture medium containing hygromycin, the transformed callus is transferred to a differentiation culture medium containing hygromycin to regenerate a plant after 28-30 days of culture, the regenerated plant is sampled to extract DNA, Taqman detects MAD7 positive single plant, and amplification sequencing is carried out on a target site of the positive single plant. The results show that the system can efficiently cut multiple targets of multiple endogenous genes in rice cells, the editing efficiency is as high as 59.6-96.9%, the frequency (including homo) of editing two alleles at the same time is as high as 34.8-95.9%, and the nucleic acid construct or the combination CRISPR-MAD7 constructed according to the invention can be efficiently edited in plant cells (Table 2).

Table 2 PAM-sg sequence of single-site knockout of multiple endogenous genes of rice in CRISPR/MAD7 (ncNLS) system and identification result of T0 generation plant

Note: the number in brackets is the number of single plants without sequencing results; the PAM sequence is underlined; WT wild type, He heterozygous mutant, Bi allelic double mutant (comprising Homo homozygous mutant). Editing efficiency (%) = number of mutants/(MAD 7)⁺Positive individual-number of failed sequenced strains) 100; biallelic frequency (%) = number of Biallelic strains/(MAD 7)⁺Positive individual-number of failed sequenced strains) 100.

Example 3 Single site knockout of different NLS positions and numbers of CRISPR-MAD7 System on Rice endogenous Gene

In example 1, PCR amplification products of CRISPR-MAD7 system were ligated to the expression vector PCAMBIA-CRISPR-MAD7 via BsrGI and AvrII cleavage sites, or ApaI cleavage site, respectively, to construct a pCAMBIA-CRISPR-nNLS-MAD7 (removing NLS nuclear localization sequence at C-terminal of MAD7 nucleic acid editing enzyme) and pCAMBIA-CRISPR-MAD7-cNLS (removing NLS nuclear localization sequence at N-terminal of MAD7 nucleic acid editing enzyme) system. The constructed vector is transformed into agrobacterium EHA105, then the strain infects callus of the rice variety Nanjing 46, the callus is recovered and cultured for 4 days at 30 ℃ after 3 days of total culture, the transformed callus is transferred to a screening culture medium containing hygromycin, and the transformed callus is transferred to a differentiation culture medium containing hygromycin to regenerate plants after 28-30 days of culture. And (3) sampling regenerated plants, extracting DNA, detecting MAD7 positive individuals by Taqman, and performing amplification sequencing on target sites of the positive individuals. The results show that the editing efficiency of the CRISPR-MAD7 (ncNLS), CRISPR-MAD7 (nNLS) and CRISPR-MAD7 (cNLS) systems in rice cells is 89.9%, 89.0% and 78.7% respectively, the biallelic (containing homo) frequency is 77.5%, 85.4% and 54.7% respectively, the editing efficiency of the MAD7 protein N-terminal nuclear localization signal vector in rice cells is slightly reduced, and the frequency of biallelic editing is obviously reduced (Table 3).

Table 3 identification results of different NLS positions and numbers of CRISPR-MAD7 system on rice endogenous gene single-site knockout PAM-sg sequence and T0 generation plant

Note: the number in brackets is the number of single plants without sequencing results; the PAM sequence is underlined; WT wild type, He heterozygous mutant, Bi allelic double mutant (comprising Homo homozygous mutant). Editing efficiency (%) = number of mutants/(MAD 7)⁺Positive individual-number of failed sequenced strains) 100; biallelic frequency (%) = number of Biallelic strains/(MAD 7)⁺Positive individual-number of failed sequenced strains) 100.

Example 4 Single-site knockout of different sg lengths of CRISPR-MAD7 System on rice endogenous genes

The same sg sequence as in example 1, the length of which is respectively removed by 2bp and 4bp or increased by 2bp, and the sg sequence is connected to the pCAMBIA-CRISPR-MAD7 expression vector through AvrII and RsrII enzyme cutting sites. The constructed vector is transformed into agrobacterium EHA105, then the strain infects callus of the rice variety Nanjing 46, the callus is recovered and cultured for 4 days at 30 ℃ after 3 days of total culture, the transformed callus is transferred to a screening culture medium containing hygromycin, and the transformed callus is transferred to a differentiation culture medium containing hygromycin to regenerate plants after 28-30 days of culture. And (3) sampling regenerated plants, extracting DNA, detecting MAD7 positive individuals by Taqman, and performing amplification sequencing on target sites of the positive individuals. The results show that as the sg length is increased from 19bp, 21bp and 23bp to 25bp, the editing frequency and biallelic (homo-containing) frequency in rice cells are respectively reduced from 95.7% and 89.2% to 85.9% and 56.5%, and the editing rate and the biallelic frequency between sg vectors with different lengths are not significantly different except that the frequency of the biallelic with 25bp sg is significantly reduced compared with those of other lengths (Table 4).

Table 4 identification results of single-site knockout PAM-sg sequences and T0 generation plants of rice endogenous genes by different sg lengths of CRISPR-MAD7 system

Note: the number in brackets is the number of single plants without sequencing results; the PAM sequence is underlined; WT wild type, He heterozygous mutant, Bi allelic double mutant (comprising Homo homozygous mutant). Editing efficiency (%) = number of mutants/(MAD 7)⁺Positive individual-number of failed sequenced strains) 100; biallelic frequency (%) = number of Biallelic strains/(MAD 7)⁺Positive individual-number of failed sequenced strains) 100.

Example 5 CRISPR-MAD7 vector comprising DR-sg sequence Strand used for rice multi-site knockout

Because of the ability of MAD7 to autonomously cleave and process pre-crRNA, the editing sites of Cpf1 (OsDEP 1-g6, OsBEL260-g1, OsRoc5-g1, OsHD3A-g 22) reported in 4 articles in example 2 were selected and linked by the mature DR sequence of MAD7 under the control of the same OsU3 promoter (SEQ ID NO. 3). This expression cassette was then ligated to the MAD7 expression cassette and placed within the LB and RB sequences of pCAMBIA. The constructed vector is transformed into agrobacterium EHA105, then the strain infects callus of the rice variety Nanjing 46, the callus is recovered and cultured for 4 days at 30 ℃ after 3 days of total culture, the transformed callus is transferred to a screening culture medium containing hygromycin, and the transformed callus is transferred to a differentiation culture medium containing hygromycin to regenerate plants after 28-30 days of culture. And (3) sampling regenerated plants, extracting DNA, detecting MAD7 positive individuals by Taqman, and performing amplification sequencing on target sites of the positive individuals. The results show that the mutation frequencies of the 4 genes in the system are 34.0%, 80.9%, 3.2% and 3.2%, respectively, and the efficiency of editing two alleles simultaneously is 11.7%, 59.6%, 0% and 0%, respectively. The first gene and the second gene of the system can efficiently edit genes in rice cells, and because U3 belongs to Pol III type promoters, the capacity of driving a long chain is limited, the editing efficiency of the third gene and the fourth gene of the system is obviously reduced, and all mutant genotypes are heterozygous; no individuals with both alleles edited simultaneously and individuals with multiple genes edited simultaneously were found (Table 5).

Table 5 PAM-sg sequence knockout of different rice genes by CRISPR-MAD7 (DR tandem sgRNA) multigene editing system and identification result of T0 generation plant

Note: the number in brackets is the number of single plants without sequencing results; the PAM sequence is underlined; WT wild type, He heterozygous mutant, Bi allelic double mutant (comprising Homo homozygous mutant). Editing efficiency (%) = number of mutants/(MAD 7)⁺Positive individual-number of failed sequenced strains) 100; biallelic frequency (%) = number of Biallelic strains/(MAD 7)⁺Positive individual-number of failed sequenced strains) 100.

Example 6 CRISPR-MAD7 vector for Rice Multi-site knockout Using tRNA tandem DR-sg sequences

The DR-sg sequence of example 5 was ligated in a spacer through the recognition site of RNAase in the endogenous tRNA processing system and under the control of the same OsU3 promoter (SEQ ID NO. 4). This expression cassette was then ligated to the MAD7 expression cassette and placed within the LB and RB sequences of pCAMBIA. The constructed vector is transformed into agrobacterium EHA105, then the strain infects callus of the rice variety Nanjing 46, the callus is recovered and cultured for 4 days at 30 ℃ after 3 days of total culture, the transformed callus is transferred to a screening culture medium containing hygromycin, and the transformed callus is transferred to a differentiation culture medium containing hygromycin to regenerate plants after 28-30 days of culture. And (3) sampling regenerated plants, extracting DNA, detecting MAD7 positive individuals by Taqman, and performing amplification sequencing on target sites of the positive individuals. The results show that the mutation frequencies of the 4 genes of the system are 49.4%, 91.0%, 71.9% and 68.2% respectively, the efficiency of simultaneously editing two alleles is 14.6%, 78.7%, 58.4% and 47.7% respectively, and the simultaneous mutation frequency of the 4 genes accounts for 38.2% of that of a positive single plant, so that the system can efficiently carry out multigene editing in rice cells simultaneously (Table 6).

Table 6 PAM-sg sequence knockout of different rice genes and identification result of T0 generation plant by CRISPR-MAD7 (tRNA DR-sgRNA in series) polygene editing system

Note: the number in brackets is the number of single plants without sequencing results; lower partThe dash line represents a PAM sequence; WT wild type, He heterozygous mutant, Bi allelic double mutant (comprising Homo homozygous mutant). Editing efficiency (%) = number of mutants/(MAD 7)⁺Positive individual-number of failed sequenced strains) 100; biallelic frequency (%) = number of Biallelic strains/(MAD 7)⁺Positive individual-number of failed sequenced strains) 100.

Example 7 use of CRISPR-MAD7 vector for Rice Multi-site knockout with miniOsU3/miniOsU6 driving DR-sg sequences, respectively

The DR-sg sequence series of example 5 were separately driven using miniOsU3/miniOsU6 (SEQ ID NO. 5). This expression cassette was then ligated to the MAD7 expression cassette and placed within the LB and RB sequences of pCAMBIA. The constructed vector is transformed into agrobacterium EHA105, then the strain infects callus of the rice variety Nanjing 46, the callus is recovered and cultured for 4 days at 30 ℃ after 3 days of total culture, the transformed callus is transferred to a screening culture medium containing hygromycin, and the transformed callus is transferred to a differentiation culture medium containing hygromycin to regenerate plants after 28-30 days of culture. And (3) sampling regenerated plants, extracting DNA, detecting MAD7 positive individuals by Taqman, and performing amplification sequencing on target sites of the positive individuals. The results show that the system can efficiently carry out multi-gene editing in rice cells at the same time, the mutation frequencies of 4 genes are respectively 44.4%, 94.4%, 92.2% and 90.0%, the mutation frequencies of two alleles are respectively 22.2%, 93.3%, 86.7% and 87.8%, and the mutation frequencies of 4 genes account for 42.2% of positive individuals, and the system can efficiently carry out multi-gene editing in rice cells at the same time (Table 7).

TABLE 7 CRISPR-MAD7 (miniOsU 3/miniOsU6 drive DR-sgRNA respectively) polygene editing system for knocking out PAM-sg sequence and T0 generation plant identification result of different rice genes

Note: the number in brackets is the number of single plants without sequencing results; the PAM sequence is underlined; WT wild type, He heterozygous mutant, Bi allelic double mutant (comprising Homo homozygous mutant). Editing efficiency (%) = number of mutants/(MAD 7)⁺Positive Individual-sequencingNumber of failed plants) 100; biallelic frequency (%) = number of Biallelic strains/(MAD 7)⁺Positive individual-number of failed sequenced strains) 100.

Example 8 use of CRISPR-MAD7 vector driven by Pol II type promoter and having HH-HDV tandem DR-sg sequence for multiple site knockout of Rice

In examples 5-7, DR-guide arrays driven by OsU3 or miniOsU3/miniOsU6 promoters were used to achieve efficient knock-out of 4 loci in rice, but U3/U6 both belong to Pol III promoters, and have limited ability to drive long chains, and Pol III promoters do not have condition-specific or tissue-specific activation ability, but Pol II-like promoters can effectively overcome the above-mentioned drawbacks. In this example, a Pol II type promoter, maize Ubiquitin, was constructed to drive the crRNA expression cassette, two ribozymes hammerhead ribozyme (HH) and Hepatitis deltavirus ribozyme (HDV) with RNA self-cleaving activity were used to isolate the transcribed DR-guide targeting sequence (SEQ ID NO.6), and a OsU3 promoter control was constructed to drive the crRNA expression cassette, which was cloned into pCAMBIA expression vector separately from the original MAD7 expression cassette. The constructed vector is transformed into agrobacterium EHA105, then the strain infects callus of the rice variety Nanjing 46, the callus is recovered and cultured for 4 days at 30 ℃ after 3 days of total culture, the transformed callus is transferred to a screening culture medium containing hygromycin, and the transformed callus is transferred to a differentiation culture medium containing hygromycin to regenerate plants after 28-30 days of culture. And (3) sampling regenerated plants, extracting DNA, detecting MAD7 positive individuals by Taqman, and performing amplification sequencing on target sites of the positive individuals. The results showed that the mutation frequencies of OsU3-HH-HDV system 4 genes were 7.6%, 6.7%, 0% and 3.3%, respectively, and that all the mutant individuals were heterozygous except for the mutant individual in which one allele was simultaneously edited and detected on the second gene (Table 8). The ZmUbi-HH-HDV system has 4 gene mutation frequencies of 82.6%, 92.1%, 88.8% and 93.3%, the efficiency of simultaneous editing of two alleles of 62.8%, 84.3% and 92.1% (Table 8), the using effect even exceeds the editing efficiency of single-site knockout, and the frequency of simultaneous mutation of 4 genes accounts for 77.5% of that of a positive single strain, so that the system can carry out multigene editing in rice cells at the same time with high efficiency (Table 8).

TABLE 8 CRISPR-MAD7 (HH-HDV tandem DR-sgRNA) polygene editing system for knocking out PAM-sg sequence and identifying result of T0 generation plant

Note: the number in brackets is the number of single plants without sequencing results; the PAM sequence is underlined; WT wild type, He heterozygous mutant, Bi allelic double mutant (comprising Homo homozygous mutant). Editing efficiency (%) = number of mutants/(MAD 7)⁺Positive individual-number of failed sequenced strains) 100; biallelic frequency (%) = number of Biallelic strains/(MAD 7)⁺Positive individual-number of failed sequenced strains) 100.

Example 9 Single site knockout of maize endogenous genes Using the CRISPR-MAD7 (ncNLS) System

To further verify the efficiency of targeted cleavage of the CRISPR-MAD7 (ncNLS) system in plant cells, an editing site (glossy 2, Zm00001d 002353) of Cpf1 in corn reported in the article is selected, DR-sgRNA is artificially synthesized, and an overlap PCR amplification product is connected to a pCAMBIA-CRISPR-MAD7 expression vector through an ApaI enzyme cutting site. The constructed vector is transformed into agrobacterium LBA4404, then the strain infects young embryo of corn variety B104, after 7 days of total culture, the young embryo is recovered and cultured for two weeks at 28 ℃, the transformed young embryo is transferred to a screening culture medium containing mannose, after two weeks of culture, the young embryo is transferred to a differentiation culture medium containing mannose to regenerate a plant, the regenerated plant is sampled to extract DNA, Taqman detects MAD7 positive single plant, and the amplification sequencing is carried out on the target site of the positive single plant. The result shows that the system can cut in a targeted manner in corn cells to generate mutation and generate mutant plants, and the editing efficiency is 6.5%; when a base C is added at the 5' end of the mature DR sequence of the MAD7 (FIG. 14), the efficiency of target cutting mutation generation of the MAD7 in corn cells is improved, the editing efficiency is improved to 13.7%, and a new idea is provided for editing and applying the MAD7 in corn.

TABLE 9 PAM-sg sequence of single-site knockout of maize endogenous gene in CRISPR/MAD7 (ncNLS) system and identification result of T0 generation plant

Note: the number of the carrier is in parentheses; the PAM sequence is underlined; editing efficiency (%) = number of mutants/(MAD 7)⁺Positive individual-number of failed sequenced strains) 100; biallelic frequency (%) = number of Biallelic strains/(MAD 7)⁺Positive individual-number of failed sequenced strains) 100.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> family Ji Daron (Beijing) Biotechnology Ltd

<120> MAD7-NLS fusion protein, nucleic acid construct for site-directed editing of plant genome and application thereof

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3792

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgaacaatg gcaccaacaa tttccagaat ttcatcggca tctccagcct gcaaaagaca 60

ctcaggaacg ccctgatccc gacggagaca acacagcagt tcatcgtgaa gaatggcatc 120

atcaaggagg atgagctccg gggcgagaac cgccagatcc tgaaggacat catggacgat 180

tactaccggg gcttcatctc cgagacactg tcttcaatcg acgatatcga ctggacgagc 240

ctcttcgaga agatggagat ccagctgaag aatggcgata acaaggacac actcatcaag 300

gagcagacgg agtaccgcaa ggccatccat aagaagttcg cgaatgacga taggttcaag 360

aacatgttct cggcgaagct catctccgat atcctgccag agttcgtgat ccacaacaat 420

aactactcag cctcggagaa ggaggagaag acccaggtca tcaagctgtt ctcccggttc 480

gcgacatctt tcaaggacta cttcaagaat cgcgccaact gcttctctgc ggacgatatc 540

tcgtccagct cttgccatcg catcgtgaat gataacgccg agatcttctt ctcaaacgcg 600

ctcgtgtacc gcaggatcgt caagtccctg agcaatgacg atatcaacaa gatctcaggc 660

gatatgaagg actcactcaa ggagatgtcg ctggaggaga tctactcgta cgagaagtac 720

ggcgagttca tcacccagga gggcatcagc ttctacaacg acatctgcgg caaggtcaat 780

tctttcatga acctgtactg ccagaagaat aaggagaata agaacctcta caagctgcag 840

aagctccata agcagatcct ctgcatcgcc gatacaagct acgaggtgcc ttacaagttc 900

gagtccgacg aggaggtgta ccagagcgtc aatggcttcc tcgataacat ctcatcgaag 960

cacatcgtcg agaggctgcg gaagatcggc gataattaca acggctacaa cctcgacaag 1020

atctacatcg tgtccaagtt ctacgagtct gtctcacaga agacctacag ggattgggag 1080

acaatcaaca cagcgctcga gatccactac aataacatcc tgccgggcaa cggcaagagc 1140

aaggccgata aggtgaagaa ggcggtcaag aatgacctcc agaagtctat caccgagatc 1200

aatgagctcg tgtcaaacta caagctgtgc tcggacgata acatcaaggc cgagacatac 1260

atccacgaga tctcccatat cctcaataac ttcgaggcgc aggagctgaa gtacaatccg 1320

gagatccatc tcgtcgagtc cgagctgaag gccagcgagc tcaagaatgt gctggacgtc 1380

atcatgaacg cgttccactg gtgctccgtg ttcatgaccg aggagctcgt cgacaaggat 1440

aataacttct acgccgagct ggaggagatc tacgatgaga tctacccagt gatctcgctg 1500

tacaatctcg tgcgcaacta cgtcacccag aagccatact ccacaaagaa gatcaagctc 1560

aacttcggca tccctacact ggccgacggc tggtcgaagt ccaaggagta ctcgaataac 1620

gcgatcatcc tcatgaggga taatctgtac tacctcggca tcttcaatgc gaagaacaag 1680

ccagacaaga agatcatcga gggcaatacg tccgagaaca agggcgatta caagaagatg 1740

atctacaatc tcctgccggg cccaaacaag atgatcccga aggtgttcct gtccagcaag 1800

acaggcgtcg agacatacaa gccatccgcc tacatcctcg agggctacaa gcagaacaag 1860

cacatcaagt cttcaaagga cttcgatatc acgttctgcc atgatctcat cgactacttc 1920

aagaattgca tcgcgatcca ccctgagtgg aagaacttcg gcttcgattt ctcagacaca 1980

tcgacgtacg aggacatctc cggcttctac cgggaggtgg agctccaggg ctacaagatc 2040

gattggacct acatcagcga gaaggacatc gatctcctgc aggagaaggg ccagctctac 2100

ctgttccaga tctacaacaa ggatttttcc aagaagtcca cgggcaatga caacctgcat 2160

accatgtacc tgaagaatct cttcagcgag gagaacctca aggacatcgt cctcaagctg 2220

aacggcgagg ccgagatctt cttcaggaag tcgtccatca agaatccaat catccacaag 2280

aagggctcta tcctggtgaa caggacctac gaggcggagg agaaggacca gttcggcaac 2340

atccagatcg tccggaagaa tatccctgag aacatctacc aggagctcta caagtacttc 2400

aatgataagt ctgacaagga gctctcagat gaggccgcga agctgaagaa tgtggtcggc 2460

caccatgagg ccgcgacgaa catcgtgaag gattaccggt acacctacga caagtacttc 2520

ctccatatgc cgatcaccat caatttcaag gccaacaaga caggcttcat caacgaccgc 2580

atcctccagt acatcgcgaa ggagaaggat ctgcacgtca tcggcatcga ccgcggcgag 2640

aggaatctga tctacgtgtc tgtcatcgat acctgcggca acatcgtgga gcagaagtca 2700

ttcaatatcg tcaacggcta cgattaccag atcaagctca agcagcagga gggagcaagg 2760

cagattgcca ggaaggagtg gaaggagatc ggcaagatca aggagatcaa ggagggctac 2820

ctctctctcg tgatccatga gatctcaaag atggtcatca agtacaacgc catcatcgcg 2880

atggaggacc tgagctatgg cttcaagaag ggccggttca aggtggagcg ccaggtctac 2940

cagaagttcg agacaatgct catcaataag ctgaactacc tcgtgttcaa ggacatctca 3000

atcacggaga acggcggcct cctgaagggc taccagctca cctacatccc ggataagctg 3060

aagaatgtgg gccaccagtg cggctgcatc ttctacgtcc ctgccgcgta cacaagcaag 3120

atcgacccga cgaccggctt cgtgaacatc ttcaagttca aggatctgac ggtcgacgcc 3180

aagagggagt tcatcaagaa gttcgattcg atccggtacg actccgagaa gaacctcttc 3240

tgcttcacgt tcgattacaa taacttcatc acgcagaata ccgtgatgag caagagctct 3300

tggtctgtgt acacctacgg cgtccggatc aagcggcgct tcgtcaatgg ccgcttctct 3360

aacgagtcag ataccatcga catcacaaag gatatggaga agacgctgga gatgaccgac 3420

atcaactgga gggatggcca tgacctccgg caggatatca tcgactacga gatcgtgcag 3480

cacatcttcg agatcttccg cctcacagtc cagatgagga acagcctgtc tgagctcgag 3540

gaccgcgatt acgacaggct catctcacct gtgctgaatg agaataacat cttctacgat 3600

tccgcaaagg cgggcgacgc cctcccgaag gatgcggatg ccaacggcgc atactgcatt 3660

gccctgaagg gcctctacga gatcaagcag atcacggaga attggaagga ggatggcaag 3720

ttctcgcgcg acaagctgaa gatctccaat aaggattggt tcgacttcat ccagaacaag 3780

aggtacctct aa 3792

<210> 2

<211> 462

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cctaggaagg aatctttaaa catacgaaca gatcacttaa agttcttctg aagcaactta 60

aagttatcag gcatgcatgg atcttggagg aatcagatgt gcagtcaggg accatagcac 120

aagacaggcg tcttctactg gtgctaccag caaatgctgg aagccgggaa cactgggtac 180

gttggaaacc acgtgtgatg tgaaggagta agataaactg taggagaaaa gcatttcgta 240

gtgggccatg aagcctttca ggacatgtat tgcagtatgg gccggcccat tacgcaattg 300

gacgacaaca aagactagta ttagtaccac ctcggctatc cacatagatc aaagctggtt 360

taaaagagtt gtgcagatga tccgtggcag tcaaaagacc tttttaattt ctactcttgt 420

agataggaca ggtactacgt gctgatgttt ttttgcagcg ct 462

<210> 3

<211> 669

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggtctcaagg aatctttaaa catacgaaca gatcacttaa agttcttctg aagcaactta 60

aagttatcag gcatgcatgg atcttggagg aatcagatgt gcagtcaggg accatagcac 120

aagacaggcg tcttctactg gtgctaccag caaatgctgg aagccgggaa cactgggtac 180

gttggaaacc acgtgtgatg tgaaggagta agataaactg taggagaaaa gcatttcgta 240

gtgggccatg aagcctttca ggacatgtat tgcagtatgg gccggcccat tacgcaattg 300

gacgacaaca aagactagta ttagtaccac ctcggctatc cacatagatc aaagctggtt 360

taaaagagtt gtgcagatga tccgtggcag tcaaaagacc tttttaattt ctactcttgt 420

agatcagaaa gagaaggagg cacagatgtc aaaagacctt tttaatttct actcttgtag 480

atgtgccgga acagagacta catcagtcaa aagacctttt taatttctac tcttgtagat 540

taagcagctg gctgagggtg catgtcaaaa gaccttttta atttctactc ttgtagatag 600

gacaggtact acgtgctgat ggtcaaaaga cctttttaat ttctactctt gtagattttt 660

tttggtctc 669

<210> 4

<211> 1054

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cctaggaagg aatctttaaa catacgaaca gatcacttaa agttcttctg aagcaactta 60

aagttatcag gcatgcatgg atcttggagg aatcagatgt gcagtcaggg accatagcac 120

aagacaggcg tcttctactg gtgctaccag caaatgctgg aagccgggaa cactgggtac 180

gttggaaacc acgtgtgatg tgaaggagta agataaactg taggagaaaa gcatttcgta 240

gtgggccatg aagcctttca ggacatgtat tgcagtatgg gccggcccat tacgcaattg 300

gacgacaaca aagactagta ttagtaccac ctcggctatc cacatagatc aaagctggtt 360

taaaagagtt gtgcagatga tccgtggcaa acaaagcacc agtggtctag tggtagaata 420

gtaccctgcc acggtacaga cccgggttcg attcccggct ggtgcagtca aaagaccttt 480

ttaatttcta ctcttgtaga tcagaaagag aaggaggcac agataacaaa gcaccagtgg 540

tctagtggta gaatagtacc ctgccacggt acagacccgg gttcgattcc cggctggtgc 600

agtcaaaaga cctttttaat ttctactctt gtagatgtgc cggaacagag actacatcaa 660

acaaagcacc agtggtctag tggtagaata gtaccctgcc acggtacaga cccgggttcg 720

attcccggct ggtgcagtca aaagaccttt ttaatttcta ctcttgtaga ttaagcagct 780

ggctgagggt gcataacaaa gcaccagtgg tctagtggta gaatagtacc ctgccacggt 840

acagacccgg gttcgattcc cggctggtgc agtcaaaaga cctttttaat ttctactctt 900

gtagatagga caggtactac gtgctgatgg tcaaaagacc tttttaattt ctactcttgt 960

agataacaaa gcaccagtgg tctagtggta gaatagtacc ctgccacggt acagacccgg 1020

gttcgattcc cggctggtgc atttttttgg tctc 1054

<210> 5

<211> 1048

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cctaggggcc caatcggcag caaaggagtg gcccatgaag cccatcagga catgtattgc 60

agtatgggcc ggcccattac gcaattggac gacaacaaag gctagtatta gtaccacctc 120

ggctatccac atagatcaaa gctggtttaa aagagttgtg cagatgatcc gtggcagtca 180

aaagaccttt ttaatttcta ctcttgtaga tcagaaagag aaggaggcac agattttttt 240

tggcccaatc ggcagcaaag gaggcccagg acggaatagg ggaaaaagtt ggcccgatag 300

gagggaaagg cccaggtgct tacgtgcgag gtaggcctgg gctctcagca cttcgattcg 360

ttggcaccgg ggtaggatgc aatagagagc aacgtttagt accacctcgc ttagctagag 420

caaactggac tgccttatat gcgcgggtgc tggcttggct gccggtcaaa agaccttttt 480

aatttctact cttgtagatg tgccggaaca gagactacat catttttttg gcccaatcgg 540

cagcaaagga ggcccacgag cgtgtactac ggcccgggat gccgctgggc gctgcggggg 600

ccgttggatg gggatcggtg ggtcgcggga gcgttgaggg gagacaggtt tagtaccacc 660

tcgcctaccg aacaatgaag aacccacctt ataaccccgc gcgctgccgc ttgtgttggt 720

caaaagacct ttttaatttc tactcttgta gattaagcag ctggctgagg gtgcattttt 780

tttggcccaa tcggcagcaa aggaggccca cttttctccg tggtgggccg atccagctag 840

aggtccggcc cacaagtggc ccttgccccg tgggacggtg ggattgcaga gcgcgtgggc 900

ggaaacaaca gtttagtacc acctcgctca cgcaacgacg cgaccacttg cttataagct 960

gctgcgctga ggctcaggtc aaaagacctt tttaatttct actcttgtag ataggacagg 1020

tactacgtgc tgatgttttt ttggtctc 1048

<210> 6

<211> 725

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggtctctttg acctgatgag tccgtgagga cgaaacgagt aagctcgtcg tcaaaagacc 60

tttttaattt ctactcttgt agatcagaaa gagaaggagg cacagatggc cggcatggtc 120

ccagcctcct cgctggcgcc ggctgggcaa catgcttcgg catggcgaat gggacgaata 180

cgacctttga cctgatgagt ccgtgaggac gaaacgagta agctcgtcgt caaaagacct 240

ttttaatttc tactcttgta gatgtgccgg aacagagact acatcaggcc ggcatggtcc 300

cagcctcctc gctggcgccg gctgggcaac atgcttcggc atggcgaatg ggacgaatac 360

gacctttgac ctgatgagtc cgtgaggacg aaacgagtaa gctcgtcgtc aaaagacctt 420

tttaatttct actcttgtag attaagcagc tggctgaggg tgcatggccg gcatggtccc 480

agcctcctcg ctggcgccgg ctgggcaaca tgcttcggca tggcgaatgg gacgaatacg 540

acctttgacc tgatgagtcc gtgaggacga aacgagtaag ctcgtcgtca aaagaccttt 600

ttaatttcta ctcttgtaga taggacaggt actacgtgct gatgggccgg catggtccca 660

gcctcctcgc tggcgccggc tgggcaacat gcttcggcat ggcgaatggg actttttttg 720

gtctc 725

<210> 7

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tttaaggaca ggtactacgt gctgatg 27

<210> 8

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tttggaagga cagttaggca gcctggg 27

<210> 9

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tttccagaaa gagaaggagg cacagat 27

<210> 10

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tttgccaaca tacagattat agattaa 27

<210> 11

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tttggtgccg gaacagagac tacatca 27

<210> 12

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tttgtaagca gctggctgag ggtgcat 27

<210> 13

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tttaatgcat ccagaacgag aaacggg 27

<210> 14

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tttcagtgtc actccgtcca acccatt 27

<210> 15

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tttgctgtca ctgtagcaga ggacatg 27

<210> 16

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tttggaatat aatgatgctt gagcctt 27

<210> 17

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tttaaggaca ggtactacgt gct 23

<210> 18

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tttaaggaca ggtactacgt gctga 25

<210> 19

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

tttaaggaca ggtactacgt gctgatgat 29

<210> 20

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tttggtcaca gatcacaaac ttcaaat 27

<210> 21

<211> 841

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gggcccgaat tccatctaag tatcttggta aagcatggat taatttggat gcccacttca 60

ggtctatgca gctccggtgc cttgtgattg tgagttgtga ccgatgctca tgctattctg 120

catttctgcg atgtatgtag ctagtagatc ttcaaaacta acaccgcatg ccatcatcat 180

ccactgcttg attttagtct caccgctggc caaaaatgtg atgatgccag aaacctcaac 240

taccttgaat caacacgggc ccaacagtgt gatgacgaca gaaacaaaaa aaaatgagcc 300

aatagttcag aaggaggcac tatgcagaaa ctacatttct gaaggtgact aaaaggtgag 360

cgtagagtgt aattactagt agtttagcca ccattaccca aatgctttcg agcttgtatt 420

aagatttcct aagctgagca tcatcactga tctgcaggcc accctcgctt cgctgccaag 480

atcaacagca accatgtggc ggcaacatcc agcattgcac atgggctaaa gattgagctt 540

tgtgcctcgt ctagggatca gctgaggtta tcagtctttc ctttttttca tccaggtgag 600

gcatcaagct actactgcct cgattggctg gacccgaagc ccacatgtag gataccagaa 660

tgggccgacc caggacgcag tatgttggcc agtcccaccg gttagtgcca tctcggttgc 720

tcacatgcgt agaagccagc ttaaaaattt agctttggtg actcacagca gtcaaaagac 780

ctttttaatt tctactcttg tagatgtcac agatcacaaa cttcaaattt tttttgggcc 840

c 841

Claims (14)

1. A MAD7-NLS fusion protein, which has the following structure:

B1-C-B2, B1-C or C-B2;

wherein,

c is MAD7 protein;

b1 and B2 are independent nuclear localization signal sequences.

2. The MAD7-NLS fusion protein of claim 1, wherein said nuclear localization signal sequence is selected from the group consisting of: one of SV40, KRP2, MDM2, CDc25C, DPP9, MTA1, CBP80, AreA, M9, Rev, hTAP, MyRF, EBNA-6, TERT, or Tfam, or a combination of any two or more thereof.

3. The MAD7-NLS fusion protein according to claim 1 or 2, wherein the MAD7-NLS fusion protein further comprises a signal peptide and/or a protein tag sequence at the N-terminus.

4. A nucleic acid construct for site-directed editing of a plant genome comprising a first expression cassette comprising, in sequential linkage, a first promoter, a nucleotide sequence encoding the MAD7-NLS fusion protein of any one of claims 1-3, and a first terminator.

5. The nucleic acid construct of claim 4, wherein the first promoter is a Pol II type promoter selected from the group consisting of Ubi, Actin, CmYLCV, UBQ, 35S, SPL, one or a combination of any two of the tissue specific promoters YAO, CDC45, rbcS and the inducible promoter XEV.

6. The nucleic acid construct of claim 4, further comprising a second expression cassette comprising a second promoter, a plurality of tandem repeats, connected in sequence;

the repetitive sequence is one or two of a mature direct repetitive sequence and an immature direct repetitive sequence.

7. The nucleic acid construct of claim 6, wherein the second promoter is a Pol II type or Pol III type promoter; the second promoter is selected from one, two or more of OsU3, OsU6a, OsU6b, OsU6c, Actin, 35S, Ubi, UBQ, SPL, CmYLCV, tissue specific promoter YAO, CDC45, rbcS or inducible promoter XEV.

8. The nucleic acid construct of claim 6, wherein the second expression cassette further comprises a second termination sequence linked to the end of the repeat sequence; the second termination sequence is selected from polyT, NOS, polyA or a combination thereof.

9. The nucleic acid construct of claim 6, wherein the repeat sequence further comprises a target site leader sequence sg; the length of the target site guide sequence sg is 17-35bp and/or the number of repeated sequences is 2-50.

10. The nucleic acid construct of claim 9, wherein the nucleic acid construct is one or more selected from the group consisting of SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, and SEQ ID No. 21.

11. The nucleic acid construct of any one of claims 4-10, wherein the nucleic acid construct is a vector comprising both the first expression cassette and the second expression cassette, or,

a vector combination consisting of a first vector comprising the first expression cassette and a second vector comprising the second expression cassette, respectively.

12. A kit for gene editing in a plant comprising the nucleic acid construct of any one of claims 4-11.

13. The kit of claim 12, further comprising an auxiliary vector carrying the donor DNA expression cassette.

14. A method for gene editing in a plant, comprising:

(i) introducing the nucleic acid construct of any one of claims 4-11 and optionally a donor nucleic acid fragment into a plant cell, plant tissue or plant body and performing gene editing in said plant cell, plant tissue or plant body;

(ii) screening and identifying plant cells, plant tissues or plant bodies in which said gene editing has occurred;

(iii) (iii) regenerating or culturing the plant cells, plant tissues or plant bodies identified in step (ii) as having undergone said gene editing;

the plant is selected from any one of gramineae, leguminous plants, solanaceae or cruciferae plants.

Images