WO2018098935A1

WO2018098935A1 - Vector for plant genome site-directed base substitution

Info

Publication number: WO2018098935A1
Application number: PCT/CN2017/077133
Authority: WO
Inventors: 朱健康; 陆钰明
Original assignee: 中国科学院上海生命科学研究院
Priority date: 2016-12-02
Filing date: 2017-03-17
Publication date: 2018-06-07
Also published as: CN106609282A

Abstract

Disclosed are a nucleic acid construct having a particular structure, and a method for a base mutation or a base substitution on a particular site of a plant genome using the construct. The nucleic acid construct comprises a first expression cassette, a second expression cassette and an optional third expression cassette, wherein the first expression cassette is an expression cassette for expressing gRNAs, the second expression cassette is an expression cassette for expressing a fusion protein comprising a CAS9 and cytosine deaminase, and the third expression cassette is an expression cassette for expressing a selection marker.

Description

Vector for site-based base substitution of plant genome

Technical field

The present invention relates to the field of biotechnology, and in particular to a vector for site-based base substitution of a plant genome and a method for site-based base substitution of a plant genome.

Background technique

Zinc finger nuclease (ZFN), transcription activator-like (TAL) effector nucleases (Talen) and CRISPR/Cas9 technologies are several breakthroughs in the field of genome editing in recent years. Sexual techniques ^1-3 . All three techniques can generate double-strand breaks in a specific site-specific cleavage DNA in an organism's genome, thereby performing site-directed editing using non-homologous end joining or homologous recombination of the organism. The ZFN and Talen technologies are guided by specific proteins, the construction of protein components is relatively complicated, and the editing efficiency needs to be improved. The CRISPR/Cas9 technology is guided by RNA, which is simple to construct in vitro and has high editing efficiency. Currently, three techniques have been successfully applied to a variety of organisms, including E. coli, yeast, rice, Arabidopsis, soybean, corn, and other ^four mice and human cells.

After the genome editing technique introduces a double-strand break at a given site in the genome of an organism, the result of editing is a random base insertion or deletion (Indel) ⁴ . So far, accurate base substitution in plant genomes has been a difficult problem. The main way to achieve this is homologous recombination, which is to achieve base substitution by exogenous donor DNA, but its efficiency is very low. Not yet effectively applied to plant research and breeding. Several existing reports on base substitutions in top international journals are only transient expression experiments ^5,6 , or the editing site itself is the screening tag ⁷ , such as the target gene of the herbicide bispyribyl acetate, lactate synthase. ALS, etc. ⁸ . On the other hand, large-scale gene sequencing indicates that most of the important agronomic trait loci are single base changes ^9,10 . Therefore, in the field of plant research and breeding, there is an urgent need for a method capable of efficient base substitution.

Therefore, there is an urgent need in the art to establish a set of efficient site-selective base replacement tools suitable for plant cells in the field of plants, and to obtain stable plants, thereby being effectively applied to plant research and breeding.

Summary of the invention

The object of the present invention is to provide an efficient fixed-point base replacement tool suitable for plant cells. And stable plants can be obtained, which can be effectively applied to plant research and breeding.

A first aspect of the invention provides a nucleic acid construct for genetically editing a plant, the nucleic acid construct comprising:

First expression cassette;

a second expression cassette; and

An optional third expression cassette;

Wherein the first expression cassette is a gRNA expression cassette for expressing gRNA;

The second expression cassette is a fusion protein expression cassette for expression of a fusion protein having the structure of Formula Ia or Ib:

A-L1-B-L2-C (Ia)

B-L1-A-L2-C (Ia)

In various styles,

A is a cytosine deaminase;

L1 is no or the first linker peptide (such as Xten)

B is Cas9 without cleavage activity or Cas9 having only single-strand cleavage activity;

L2 is a no or second linker peptide (such as Xten);

C is no or Uracil DNA glycosylase inhibitor (UGI);

The third expression cassette is a selection marker expression cassette for expressing a selection marker;

And, the first expression cassette, the second expression cassette, and the third expression cassette are each independently located on the same or different carriers.

In another preferred embodiment, the first expression cassette has the structure of formula A:

X1-X2-X3 (A)

In the formula,

X1 is a plant promoter, preferably a plant polymerase III driven promoter (such as U6, U3 promoter);

X2 is the coding sequence of gRNA;

X3 is polyT.

In another preferred embodiment, the second expression cassette has the structure of formula B:

Y1-Y2-Y3 (B)

In the formula,

Y1 is a plant promoter, preferably UBI, CaMV35S, Actin promoter;

Y2 is the coding sequence of a fusion protein having the structure of formula Ia or Ib as described above;

Y3 is a terminator.

In another preferred embodiment, the third expression cassette has the structure of formula C:

Z1-Z2-Z3 (C)

In the formula,

Z1 is a plant promoter, preferably a 35S, UBI, Actin promoter;

Z2 is a coding sequence encoding a screening marker;

Z3 is the terminator.

In another preferred embodiment, the selection marker is selected from the group consisting of a hygromycin resistance gene (HYG), a G418 and kanamycin resistance gene (NPTII), and a Basta resistance gene (BAR). , puromycin resistance gene (PAC), neomycin resistance gene (NEO), or a combination thereof.

In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same carrier.

In another preferred embodiment, the first expression cassette, the second expression cassette, and the third expression cassette are located on the same carrier.

In another preferred embodiment, the second expression cassette and the third expression cassette are located on the same carrier.

In another preferred embodiment, the vector is an expression vector that can be transfected or transformed into a plant cell.

In another preferred embodiment, the carrier is an Agrobacterium Ti carrier.

In another preferred embodiment, when two or three of the first expression cassette, the second expression cassette, and the third expression cassette are located on the same vector, the two or three expression cassettes are arranged in clusters. Thereby an expression cassette is constructed.

In another preferred embodiment, the RB sequence is contained outside the 5' end of the expression cassette, and the LB sequence is contained outside the 3' end of the expression cassette.

In another preferred embodiment, in the expression cassette, the order of arrangement of each expression cassette (from 5'-3') is selected from the group consisting of:

a first expression cassette and a second expression cassette;

a first expression cassette, a second expression cassette, and a third expression cassette;

a second expression cassette, a first expression cassette, and a third expression cassette;

a third expression cassette, a second expression cassette, and a first expression cassette;

a third expression cassette, a first expression cassette, and a second expression cassette;

a third expression cassette, a second expression cassette;

a second expression cassette, a third expression cassette.

In another preferred embodiment, the first expression cassette and the second expression cassette are located on different vectors.

In another preferred embodiment, when a single expression cassette is located on a vector, the RB sequence is contained outside the 5' end of the expression cassette; and the LB sequence is contained outside the 3' end of the expression cassette.

A second aspect of the invention provides an expression vector comprising the nucleic acid construct of the first aspect of the invention, wherein the first expression cassette, the second expression cassette, and the third expression cassette They are each independently located on the same or different carriers.

In another preferred embodiment, the expression vector backbone is pCamiba1300.

In another preferred embodiment, the carrier is an Agrobacterium Ti carrier.

In another preferred embodiment, the carrier is cyclic or linear.

A third aspect of the invention provides a host cell comprising the expression vector of the second aspect of the invention.

In another preferred embodiment, the host cell is Agrobacterium.

In another preferred embodiment, the host cell is a plant cell.

A fourth aspect of the invention provides a genetically engineered cell comprising the construct of the first aspect of the invention, or a genome thereof integrated with one or more of the constructs of the first aspect of the invention.

In another preferred embodiment, the cell is a plant cell.

In another preferred embodiment, the plant is selected from the group consisting of a gramineous plant, a leguminous plant, a cruciferous plant, or a combination thereof.

In another preferred embodiment, the plant is selected from the group consisting of rice, soybean, tomato, corn, tobacco, wheat, sorghum, Arabidopsis thaliana, barley, oats, millet, peanuts, or combinations thereof.

In another preferred embodiment, the genetically engineered cell is introduced into the cell by the method according to the first aspect of the invention by a method selected from the group consisting of Agrobacterium transformation, gene gun, microinjection, and electric shock. Method, ultrasonic method, polyethylene glycol (PEG) mediated method, or a combination thereof.

A fifth aspect of the invention provides a method for genetically editing a plant, comprising the steps of:

(i) providing the plant to be edited;

(ii) introducing the expression vector of the second aspect of the invention into the plant to be edited.

In another preferred embodiment, the introduction is introduced by Agrobacterium.

In another preferred embodiment, the introduction is by a gene gun.

In another preferred embodiment, the gene is edited as a fixed-point base substitution.

In another preferred embodiment, the site-directed mutagenesis comprises mutating C to T or C to G

In another preferred embodiment, the site-directed mutagenesis comprises site-directed mutagenesis of C in positions 4-8 of the distal end of the PAM.

A sixth aspect of the invention provides a method of preparing a transgenic plant cell, comprising the steps of:

(i) transfecting the construct of the first aspect of the invention, or the vector of the second aspect of the invention, with a plant cell such that the construct is site-directed with the chromosome in the plant cell, thereby producing The transgenic plant cell.

In another preferred embodiment, the transfection is performed using an Agrobacterium transformation method or a gene gun bombardment method.

A seventh aspect of the invention provides a method of preparing a transgenic plant cell, comprising the steps of:

(i) transfecting the construct of the first aspect of the invention, or the vector of the second aspect of the invention, with a plant cell such that the plant cell contains the construct, thereby producing the transgenic plant cell.

An eighth aspect of the invention provides a method of preparing a transgenic plant, comprising the steps of:

The transgenic plant cell prepared by the method of the sixth aspect of the invention or the method of the seventh aspect of the invention is regenerated into a plant body, thereby obtaining the transgenic plant.

A ninth aspect of the invention provides a transgenic plant, which is prepared by the method of the eighth aspect of the invention, or the plant cell of the plant comprises the construct of the first aspect of the invention or The genome is integrated with one or more of the constructs of the first aspect of the invention.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Figure 1 is a schematic diagram of site-based base substitution based on cytosine deaminase. The DNA sequence expressing Cas is ligated to a DNA sequence expressing cytosine deaminase, transformed into a plant receptor, and the DNA in the transformed plant cell expresses the fusion protein and guide RNA. The Cas protein (101) fused with cytosine deaminase (103), under the guidance of its guide RNA (102), deamination of cytosine C at the target position in the plant genome (104) to uracil U, then in plants Repair the U to T or G by the cell's repair system.

Figure 2 uses a fixed-base replacement vector of APOBEC1 and CRISPR/Cas9. (A) Schematic representation of the vector pCSGAPO1. (B) Schematic diagram of the deamination region.

Figure 3. Results of fixed-base substitution of rice callus. (A) Schematic diagram of base substitution of rice NRT1.1B gene. (B) Schematic diagram of base substitution of rice SLR1 gene. (C) High-throughput sequencing results.

Figure 4. Base substitution results of the NRT1.1B gene in rice transgenic plants. (A) Sanger sequencing results of Indel plants (#35) and base replacement plants (#15). (B) Single colony sequencing results of PCR products of base replacement plants (#15).

Figure 5 shows the results of base substitution of the SLR1 gene in rice transgenic plants. (A) Schematic and results of StyI digestion assay. (B) Sanger sequencing results of Indel plants (#07) and base substitution plants (#07, #10, #18, #23, #28, and #37). (C) Single colony sequencing results of base replacement plants (#07). (D) T0 generation plant phenotype of wild type (WT), knockout (SLR#09) and base substitution (SLR#07) of SLR1 gene.

detailed description

The present inventors have extensively and intensively studied to screen out optimized promoters for specific sites by extensive screening, and by using a nucleic acid construct of a specific structure, the present invention successfully achieves RNA-directed bases in plants for the first time. Site-directed mutagenesis (such as C mutation to T or G), and mutation efficiency can be as high as 11.5%, and up to 60% of cells in a mutant plant have been replaced.

Specifically, the present invention finds for the first time that cytosine deaminase can cleave the cytosine on the target genome of a plant cell under the guidance of CRISPR/Cas9, mutate it to T or G, and complete on this basis. The invention has been made.

As used herein, the terms "base substitution", base mutation, are used interchangeably and refer to the mutation of a base to another base, such as a C mutation to T or a C mutation to G.

As used herein, "screening marker gene" refers to a gene used for screening a transgenic cell or a transgenic animal in a transgenic process, and the screening marker gene useful in the present application is not particularly limited, and includes various screening marker genes commonly used in the transgenic field, representative examples. Including (but not limited to): hygromycin resistance gene (Hyg), kanamycin resistance gene (NPTII), neomycin gene, puromycin resistance gene, hygromycin resistance gene (HYG , G418 and kanamycin resistance gene (NPTII), Basta resistance gene (BAR), puromycin resistance gene (PAC), and/or neomycin resistance gene (NEO).

As used herein, the term "expression cassette" refers to a stretch of polynucleotide sequences comprising a gene to be expressed and a sequence component that expresses the desired element. For example, in the present invention, the term "screening marker expression cassette" refers to a polynucleotide sequence comprising a sequence encoding a selection marker and a sequence component expressing the desired element. The components required for expression include a promoter and a polyadenylation signal sequence. In addition, the selection marker expression cassette may or may not contain other sequences including, but not limited to, enhancers, secretion signal peptide sequences, and the like.

In the present invention, the promoter suitable for the exogenous gene expression cassette and the selection marker gene expression cassette may be any of the common promoters, and it may be a constitutive promoter or an inducible promoter. Preferably, the promoter is a constitutive promoter, such as a UBI promoter, and other plant promoters suitable for eukaryotic expression.

The term "plant promoter" as used herein 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 a plant, a microorganism (such as a bacterium, a virus) or an animal, or a synthetic or engineered promoter.

As used herein, the term "plant terminator" refers to a terminator capable of stopping transcription in a plant cell. The plant transcription terminator may be derived from a plant, a microorganism (such as a bacterium, a virus) or an animal, or a synthetic or engineered terminator. Representative examples include (but are not limited to): Nos terminator.

The term "Cas protein" as used herein refers to a nuclease. A preferred Cas protein is the Cas9 protein. Typical Cas9 proteins include, but are not limited to, Cas9 derived from Streptococcus pyogenes. In the present invention, the Cas9 protein is a mutated Cas9 protein, specifically, a mutant Cas9 protein having no cleavage activity or only single-strand cleavage activity.

As used herein, the term "coding sequence of a Cas protein" refers to a nucleotide sequence that encodes a Cas protein. In the case where the inserted polynucleotide sequence is transcribed and translated to produce a functional Cas protein, the skilled artisan will recognize that because of the degeneracy of the codon, a large number of polynucleotide sequences can encode the same polypeptide. . In addition, the skilled person will also recognize that different species have a certain preference for codons, and may optimize the codons of the Cas protein according to the needs of expression in different species. These variants are all referred to by the term "Cas protein. The coding sequence is specifically covered. Furthermore, the term specifically encompasses a full-length sequence substantially identical to the Cas gene sequence, as well as a sequence encoding a protein that retains the function of the Cas protein.

As used herein, the term "plant" includes whole plants, plant organs (such as leaves, stems, roots, etc.), seeds and plant cells, and progeny thereof. The kind of plant which can be used in the method of the present invention is not particularly limited, and generally includes any higher plant type which can be subjected to transformation techniques, including monocots, dicots, and gymnosperms.

Construct of the invention

In the present invention, a nucleic acid construct for gene editing of a plant is provided, the nucleic acid construct comprising:

First expression cassette;

a second expression cassette; and

An optional third expression cassette;

A-L1-B-L2-C (Ia)

B-L1-A-L2-C (Ia)

In various styles,

A is a cytosine deaminase;

L1 is no or the first linker peptide (such as Xten)

L2 is a no or second linker peptide (such as Xten);

C is no or Uracil DNA glycosylase inhibitor (UGI);

The various elements used in the constructs of the present invention are known in the art, and thus those skilled in the art can obtain corresponding elements by conventional methods such as PCR methods, full artificial chemical synthesis, and enzymatic cleavage methods, and then pass The well-known DNA ligation techniques are joined together to form the constructs of the present invention.

The construction of the present invention is carried out by inserting the construct of the present invention into an exogenous vector, especially a vector suitable for transgenic plant manipulation.

The transgenic plant cells are prepared by transforming the vector of the present invention into plant cells to mediate the integration of the plant cell chromosomes by the vector of the present invention.

The transgenic plant cells of the present invention are regenerated into plant bodies to obtain transgenic plants.

The above nucleic acid construct constructed by the present invention can be introduced into a plant cell by a conventional plant recombination technique (for example, Agrobacterium transfer technology) to obtain a nucleic acid construct (or a vector carrying the nucleic acid construct). Plant cells, or plant cells in the genome in which the nucleic acid construct is integrated.

Vector construction

The main feature of this vector is the linkage of cytosine deaminase to the Cas protein in the CRISPR/Cas system to form a fusion protein, which is directed by the guide RNA to a target site in the genome. Since cytosine deaminase directly deacetylates cytosine C to form U, DNA double-strand cleavage activity of Cas protein is not required. Therefore, in the present invention, the Cas protein is a mutant Cas protein having no cleavage activity. After deamination of cytosine C to form U, plant cells initiate base mismatch repair and repair U-G to T-A or C-G. In order to increase the ratio of U-G to T-A, the DNA single-strand nicking activity of the Cas protein can be retained, that is, the single-strand nicking is caused in the non-edited single strand (editing the matching strand of the target C). Therefore, preferably, the cytosine deaminase is fused to a Cas protein having single-strand nicking activity to form a fusion protein. In general, in order to increase the activity of the fusion protein, the proteins are usually linked by some flexible short peptides, namely Linker. Preferably, the Linker can use XTEN.

In order to increase the efficiency of repair, a strong promoter suitable for plant cells, such as the CaMV 35S promoter or the UBI promoter, is generally selected. The expression cassette of the guide RNA suitable for plant cells is selected and constructed in the same vector as the fusion protein expression cassette described above. Obviously, these two expression cassettes can also be on different DNA molecules.

Target design

After the cytosine deaminase is directed to the target site by the CRISPR/Cas9 system, the region of action of the deaminase is generally fixed. For example, after the cytosine deaminase APOBEC1 protein is linked to the N-terminus of Cas9 via a 16 amino acid XTEN Linker, the deamination region is the 4-8 base region of the guilde RNA, which is Amino window area" (Figure 2). According to this principle, the cytosine to be edited is in the "deamination window" when designing the target.

Genetic transformation

The above vector is introduced into a plant recipient by a suitable method. The introduction methods include, but are not limited to, gene gun method, microinjection method, electric shock method, ultrasonic method, and polyethylene glycol (PEG)-mediated method. Receptor plants include, but are not limited to, rice, soybean, tomato, corn, tobacco, wheat, sorghum, and the like. After the above DNA vector or fragment is introduced into a plant cell, the DNA in the transformed plant cell expresses the fusion protein and the guide RNA. The Cas protein fused with cytosine deaminase, under the guidance of its guide RNA, deamination of cytosine C at the target site to uracil U, and then repairs U to T or G under the action of the plant cell repair system. Further, the plants after base substitution are obtained by tissue culture.

application

The invention can be used in the field of plant genetic engineering for plant research and breeding, especially genetic improvement of economically valuable crops and forestry crops.

The main advantages of the invention include:

(1) The present invention provides, for the first time, an efficient method for realizing site-directed base mutations in plants, which can be widely used for plant research and breeding.

(2) The method of site-directed base mutation of the present invention can efficiently perform base mutation (e.g., C mutation to T or G) at a specific position of a plant cell.

(3) The effect of base mutation in plant cells by the method of the present invention is as high as 11.5%, and up to 60% of cells in one mutant plant undergo base substitution.

(4) The method of the present invention can effectively avoid random integration of foreign DNA when performing precise base substitution on plant cells without additionally introducing donor DNA.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Percentages and parts are by weight unless otherwise stated. The experimental materials and reagents referred to in the present invention can be obtained from commercially available channels unless otherwise specified.

General method

The mouse cytosine deaminase APOBEC1 was synthesized in vitro and ligated to the N-terminus of the mutated Cas9 (D10A), and the fusion protein was formed by 16aa XTEN flexible short peptide, and its nucleotide sequence is SEQ ID NO.:1. Shown as follows:

Its protein sequence is shown as SEQ ID NO.:

This sequence was constructed into the rice pCambia1300 vector (purchased from the Camiba Institute, Australia) and initiated by the plant strong promoter UBI. At the same time, the expression cassette of the guide RNA was also constructed into the vector to generate the vector pCSGAPO1 (Fig. 2A). This vector was applied to the following examples.

Example 1 Implementation of Site-Based Base Replacement in Rice Callus

First, the application of the invention was attempted to achieve base substitution in rice callus. We choose two important rice The genes NRT1.1B and SLR1. NRT1.1B can control the absorption of nutrient nitrogen in rice and increase yield; SLR1 controls the synthesis of gibberellin and affects the height of rice plants. Using the present invention, we designed corresponding targets and successfully detected fixed-point base substitutions in rice callus by high-throughput sequencing, with an efficiency of 11.5%, which is much higher than in previous plant protoplasts. Transient experiments using homologous recombination. The specific operation process is as follows:

1.1 Vector Construction and Genetic Transformation

The amino acid to be edited in rice NRT1.1B gene is Thr327, and the amino acid to be edited in SLR1 gene is Ser97. For these two sites, we designed the targets separately. As shown in Figures 3A and 3B, the cytosine C to be replaced is all within the "deamination window" of the target. Therefore, we constructed the guide RNA expression cassettes (SEQ ID NO.: 2 and SEQ ID NO.: 3) of these two targets to the vector pCSGAPO1 (obtained from Shanghai Institute of Biological Sciences, Chinese Academy of Sciences) to obtain base substitution. Vectors pCSGAPO1-NRT and pCSGAPO1-SLR1.

The above two vectors were transformed into Agrobacterium EAH105, and then the callus of Flower 11 in rice was transformed. After 4 weeks of screening culture, 100-200 resistant calli granules were selected and added to the new medium for 2 weeks. Finally, all the resistant callus granules were picked out, and the genomic DNA was extracted and mixed for base substitution detection.

1.2 base substitution efficiency test

After obtaining the total genomic DNA, primers were designed (Table 1), NRT1.1B and SLR1 were amplified by PCR, and the PCR products were subjected to high-throughput sequencing analysis. As shown in Fig. 3C, at the target position of NRT1.1B, the cytosine at position 7 was replaced by T or G with an efficiency of 1.4% and 1.6%, respectively; at the target position of SLR1, The 4th position of the guide RNA produced a C→T base substitution with an efficiency of 2.9%, while the cytosine at position 6 produced C→T and C→G base substitutions with efficiencies of 11.5% and 3.9, respectively. %. At the same time, we also detected a certain ratio of Indel, which was caused by single-strand nicking of active Cas9 (D10A). Since the targeting efficiency of guide RNA designed by NRT1.1B is lower than that of SLR1, the base substitution efficiency is also lower than that of SLR1. These experimental results show that the invention can be effectively applied to fixed-point base substitution of plant genomes.

Table 1. Primers used to amplify the target

Example 2 Obtaining Rice with Site-Related Base Replacement

The two base substitution vectors pCSGAPO1-NRT and pCSGAPO1-SLR1 in Example 1 were transformed into Agrobacterium EHA105, and 37 transgenic plants editing NRT1.1B and 45 transgenes editing SLR1 were obtained by conventional rice genetic transformation. Plant (Table 2).

Table 2. Statistics on base substitution efficiency of rice transgenic plants

For 37 transgenic plants editing NRT1.1B, we used Sanger sequencing for DNA sequencing analysis. Sequencing results showed that 4 of them produced Indel mutations at the target site, and 1 plant (NRT#15) produced C→G base substitution at the 7th position of the guide RNA. The PCR amplification product of the transgenic plant was cloned into the T vector, and 17 monoclonal clones were selected for sequencing. The results showed that the C→G base substitution efficiency in the plant was 23.5% (4/17).

For the editing of SLR1, since the C→T base substitution can generate the cleavage site of StyI, the base substitution of the site can be detected by restriction enzyme polymorphism. As shown in Figure 5A, for the C→T base-substituted PCR product (404 bp), StyI can digest it into two bands of 90 bp + 314 bp. The results showed that 6 of the 45 transgenic plants editing SLR1 produced C→T base substitution at position 6 of their guide RNA. The Sanger sequencing analysis of these 45 plants further verified the experimental results. We cloned the PCR amplification product of transgenic plant SLR#07 into T vector, and selected 10 monoclonals for sequencing. The results showed that the C→T base substitution efficiency in the plant was 60% (6/10). This result is similar to the editing result of NRT1.1B, that is, the cytosine on the SLR1 target has not been completely replaced, that is, the base substitution sites in the transgenic plants NRT#15 and SLR#07 are chimeric or Heterozygous. However, in these transgenic plants, a base substitution rate of up to 23.5%-60% at the target site is sufficient to inherit to the next generation.

According to previous studies, the base of the SLR1 gene at this site was replaced by a dominant mutation, which could affect the planting. Plant growth height. By observing the T0 generation plants of SLR1, it was found that the stems and leaves of SLR1 knockout plants were slender, and the transgenic plants with base substitutions were significantly shorter than the wild-type T0 plants, showing a short and strong phenotype. (Fig. 5D). These results further demonstrate that, with the application of the present invention, fixed-point base substitutions have been successfully performed at this site, and the substitution efficiency is high.

In this example, we have only taken conventional genetic transformation work to obtain plants with fixed-point base substitutions, indicating the great potential of the present invention for widespread application and application in plant research and breeding.

Moreover, it has been found through extensive experiments that the animal promoter cannot perform site-based base substitution on the plant of the present invention (such as rice), and only the plant promoter of the present invention (especially a rice-specific strong promoter) can be used for the plant of the present invention ( For example, rice) performs site-based base substitution, and the base substitution efficiency is up to 11.5%, and up to 60% of cells in a mutant plant have base substitutions.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

references

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3 Mahfouz, MMet al. De novo-engineered transcription activator-like effector (TALE)hybrid nuclease with novel DNA binding specificity creates double-strand breaks.Proceedings of the National Academy of Sciences of the United States of America 108,2623-2628 , doi: 10.1073/pnas.1019533108 (2011).

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Claims

B structure:

Y1-Y2-Y3 (B)

In the formula,

Y1 is a plant promoter, preferably UBI, CaMV35S, Actin promoter;

Y2 is the coding sequence of a fusion protein having the structure of formula Ia or Ib as described above;

Y3 is a terminator.
An expression vector comprising the nucleic acid construct of claim 1, wherein the first expression cassette, the second expression cassette, and the third expression cassette are each independently located in the same Or on different carriers.
A host cell comprising the expression vector of claim 4.
A genetically engineered cell, characterized in that the cell comprises the construct of claim 1, or the genome thereof is integrated with one or more of the construct of claim 1.
A method for genetically editing a plant, comprising the steps of:

(i) providing the plant to be edited;

(ii) introducing the expression vector of claim 4 into the plant to be edited.
A method for preparing a transgenic plant cell, comprising the steps of:

(i) transducing the construct of claim 1 or the vector of claim 4 into a plant cell such that the construct and site-directed mutagenesis of the chromosome in the plant cell produce the transgenic plant cell.
A method for preparing a transgenic plant cell, comprising the steps of:

(i) transducing the construct of claim 1 or the vector of claim 4 to a plant cell such that the plant cell contains the construct, thereby producing the transgenic plant cell.
A method for preparing a transgenic plant, comprising the steps of:

The transgenic plant cell prepared by the method of claim 8 or 9 is regenerated into a plant body to obtain the transgenic plant.