阅读说明：本技术 一种BSMV病毒载体介导的CRISPR/Cas9重组载体及其应用 (BSMV (human cytomegalovirus) viral vector-mediated CRISPR (clustered regularly interspaced short palindromic repeats)/Cas 9 recombinant vector and application thereof ) 是由 师恭曜 田保明 位芳 曹刚强 邵伟 翁耀毅 杨戊辰 张泽 张容嘉 郭园园 于 2020-12-18 设计创作，主要内容包括：本发明公开了一种BSMV病毒载体介导的CRISPR/Cas9重组载体,其中CRISPR/Cas9包括Cas9蛋白和sgRNA,重组载体包括BSMV介导的sgRNA重组载体和BSMV介导的split-Cas9重组载体。本发明还公开了该重组载体的构建方法,以及该重组载体在植物基因编辑中的应用。利用本发明重组载体可以在植物目的基因中产生系统性编辑结果,建立了一种研究植物基因功能的有效方法,为植物非转基因编辑提供了一个新的思路。(The invention discloses a BSMV viral vector mediated CRISPR/Cas9 recombinant vector, wherein the CRISPR/Cas9 comprises a Cas9 protein and sgRNA, and the recombinant vector comprises a BSMV mediated sgRNA recombinant vector and a BSMV mediated split-Cas9 recombinant vector. The invention also discloses a construction method of the recombinant vector and application of the recombinant vector in plant gene editing. The recombinant vector can generate systematic editing results in plant target genes, establishes an effective method for researching plant gene functions, and provides a new thought for non-transgenic editing of plants.)

1. A BSMV viral vector-mediated CRISPR/Cas9 recombinant vector, wherein CRISPR/Cas9 comprises a Cas9 protein and sgrnas, characterized in that the recombinant vector comprises a BSMV-mediated sgRNA recombinant vector and a BSMV-mediated split-Cas9 recombinant vector.

2. The BSMV viral vector-mediated CRISPR/Cas9 recombinant vector according to claim 1, wherein the BSMV-mediated sgRNA recombinant vector comprises the recombinant vector BSMV- γ: sgRNA2, BSMV- γ 1: sgRNA1, BSMV- γ 2: sgRNA1 and BSMV- γ 1: sgRNA2, BSMV- γ 2: sgRNA 2.

3. The BSMV viral vector-mediated CRISPR/Cas9 recombinant vector according to claim 1, wherein the BSMV-mediated split-Cas9 recombinant vector comprises the recombinant vector BSMV- γ 2: cas9N and BSMV- γ 2: cas 9C.

4. A construction method of a BSMV viral vector-mediated CRISPR/Cas9 recombinant vector is characterized by comprising the following steps:

s1, constructing a sgRNA Ti vector;

s11, designing an sgRNA target sequence of the NbPDS gene according to exon sequence information of the tobacco PDS gene, and analyzing to obtain sgRNA1 and sgRNA2 of two suitable enzyme cutting sites;

s12, carrying out single enzyme digestion on the pKSE401 vector by using BsaI, and then carrying out gel electrophoresis to recover an enzyme digestion product with the size of about 15 kb;

s13, inserting double-stranded DNA formed by annealing forward and reverse primers of sgRNA1 and sgRNA2 into a linearized vector of an enzyme digestion product in S12 to obtain recombinant vectors pKSE401-sgRNA1 and pKSE401-sgRNA 2;

s2, constructing a BSMV-mediated sgRNA recombinant vector;

s21, performing single enzyme digestion on the BSMV-gamma, BSMV-gamma 1 and BSMV-gamma 2 virus vectors by using ApaI, and recovering enzyme digestion products by gel electrophoresis;

s22, using Q5 high-fidelity DNA polymerase and sgRNA Ti vector as a template, amplifying sgRNA1 and sgRNA2 fragment sequences by PCR, and recovering amplification products by gel electrophoresis;

s23, connecting the linearized vector in S21 and the sgRNA fragment in S22 by a ligation method of LIC to form a recombinant vector BSMV-gamma: sgRNA2, BSMV- γ 1: sgRNA1, BSMV- γ 2: sgRNA1 and BSMV- γ 1: sgRNA2, BSMV- γ 2: sgRNA 2;

s3, constructing a BSMV-mediated split-Cas9 recombinant vector;

s31, performing single enzyme digestion on the BSMV-gamma 2 viral vector by using ApaI, and recovering an enzyme digestion product by gel electrophoresis;

s32, splitting the spCas9 from the 714(S) th amino acid, and using Q5 high-fidelity DNA polymerase and pKSE401 vector as a template to respectively amplify a Cas9N end and a Cas9C end to obtain a split-Cas9 fragment;

s33, connecting the linearized vector of the enzyme digestion product in S31 with the split-Cas9 fragment in S32 by a LIC connection method to form a recombinant vector BSMV-gamma 2: cas9N, BSMV- γ 2: cas 9C.

5. The method for constructing the BSMV viral vector-mediated CRISPR/Cas9 recombinant vector as claimed in claim 4, wherein the forward and reverse primers of sgRNA1 and sgRNA2 in S13 are:

sgRNA1 forward primer sequence: 5'-ATTGTTGGTAGTAGCGACTCCATG-3'

sgRNA1 reverse primer sequence: 5'-AAACCATGGAGTCGCTACTACCAA-3'

sgRNA2 forward primer sequence: 5'-ATTGGAGGCAAGAGATGTCCTAGG-3'

sgRNA2 reverse primer sequence: 5'-AAACCCTAGGACATCTCTTGCCTC-3' are provided.

6. The method for constructing the BSMV viral vector-mediated CRISPR/Cas9 recombinant vector as claimed in claim 4, wherein the PCR primer at the Cas9N end in S32 is:

forward primer sequence:

5’-AAGGAAGTTTAAATGGATTACAAGGACCACGA-3’

reverse primer sequence:

5’-CGGGCCAGCCACCGCCACCAGTGCTCACCTGAGCCTT-3’。

7. the method for constructing the BSMV viral vector-mediated CRISPR/Cas9 recombinant vector as claimed in claim 4, wherein the PCR primer at the Cas9C end in S32 is:

forward primer sequence:

5’-AAGGAAGTTTAAATGGGCCAGGGGGACTCGCT-3’

reverse primer sequence:

5’-CGGGCCAGCCACCGCCACCAGTTCACTTCTTCTTCTTCGCC-3’。

8. use of a BSMV viral vector mediated CRISPR/Cas9 recombinant vector according to any one of claims 1-3.

9. Use of a BSMV viral vector mediated CRISPR/Cas9 recombinant vector according to any one of claims 1-3 to deliver into nicotiana benthamiana leaves overexpressing SpCas 9.

10. Use of a BSMV viral vector mediated CRISPR/Cas9 recombinant vector according to any one of claims 1-3 in non-transgenic editing of plants.

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a BSMV (human papillomavirus) vector-mediated CRISPR/Cas9 recombinant vector and application thereof.

Background

A CRISPR/Cas9 targeted gene editing technology developed based on a bacterial CRISPR/Cas9(clustered differentiated short palindromic repeat/CRISPR associated nucleic acid 9, Cas9) immune system rapidly innovating a research method in the field of life science due to the characteristics of orientation, high efficiency and easiness in design. This technique requires only two necessary components: a Cas9 protein with nuclease activity and a small fragment of single-stranded guide RNA (sgRNA). sgrnas bind to Cas9 protein via scaffold to form Cas9 sgRNA ribonucleic acid protein complexes (RNPs). The complex can recognize a 5 '-NGG-3' PAM module sequence in a double-stranded DNA sequence and cause double helix melting of adjacent DNA, so that a 5 'guide sequence in the sgRNA and the single-stranded DNA have complementarity, if the 5' guide sequence and the single-stranded DNA are completely complementary to form an RNA-DNA stable double strand, targeting positioning is completed, two nuclease sites in the Cas9 are activated, and 3-4 nucleotides in front of the PAM site in the two single-stranded DNA are cut respectively. DNA double strand breaks can activate damage repair mechanisms. Cells either repair insertions, deletions or substitutions of bases at the DNA interface by non-homologous end joining (NHEJ) or, in the presence of homologous templates, introduce new fragments at the break using homology-mediated repair (HDR) to avoid re-cleavage of the Cas9: sgRNA complex. The mutations introduced by these repairs are exactly the targeted site editing that we need. Different genes can be targeted and edited by matching with the Cas9 protein only by replacing the first 20nt sequence in the sgRNA, so compared with Zinc Finger Nucleases (ZFNs) and transcription-activator-like nucleases (TALENs) and other genome editing technologies, the CRISPR/Cas9 genome editing technology is simpler and more convenient to operate, lower in cost, higher in editing efficiency and lower in off-target efficiency, and is widely applied to various plants at present.

Barley Streak Mosaic Virus (BSMV) is a RNA virus commonly used in VIGS and has applications in both monocots and dicots. The BSMV genome consists of three positive single-stranded RNAs, namely BSMV-alpha, beta and gamma. Previous studies have found that the BSMV three-component vector system can be used for overexpression of foreign genes, but successfully expressed genes are all small. In the early 2018, Cheuk and Houde from canada reported a new BSMV system overexpression system that reconstructed the gamma component of the BSMV genome into two subfractions, gamma 1 and gamma 2, which greatly increased the loading capacity of the system and provided insertion sites for two foreign genes. When the recombinant BSMV vector enters plant cells through agrobacterium mediation, the virus expression system mediates the synthesis of recombinant virus RNA in the plant cells, and the RNA is further replicated and then diffused to development and meristem, even to germ cell lines.

Therefore, researches on using a BSMV-mediated CRISPR/Cas9 recombinant vector and a sgRNA delivery and sgRNA + Cas9 co-delivery method in plants are problems to be solved at present.

Disclosure of Invention

The invention aims to provide a BSMV viral vector-mediated CRISPR/Cas9 recombinant vector and application thereof.

In order to solve the technical problems, the invention discloses a BSMV viral vector mediated CRISPR/Cas9 recombinant vector, wherein the CRISPR/Cas9 comprises a Cas9 protein and sgRNA, and the recombinant vector comprises a BSMV mediated sgRNA recombinant vector and a BSMV mediated split-Cas9 recombinant vector.

Further, the BSMV-mediated sgRNA recombinant vectors include the recombinant vector BSMV- γ: sgRNA2, BSMV- γ 1: sgRNA1, BSMV- γ 2: sgRNA1 and BSMV- γ 1: sgRNA2, BSMV- γ 2: sgRNA 2.

Further, the BSMV-mediated split-Cas9 recombinant vector includes the recombinant vector BSMV- γ 2: cas9N and BSMV- γ 2: cas 9C.

The invention also discloses a construction method of the BSMV viral vector-mediated CRISPR/Cas9 recombinant vector, which specifically comprises the following steps:

s1, constructing a sgRNA Ti vector;

s11, designing an sgRNA target sequence of the NbPDS gene according to exon sequence information of the tobacco PDS gene, and analyzing to obtain sgRNA1 and sgRNA2 of two suitable enzyme cutting sites;

s12, carrying out single enzyme digestion on the pKSE401 vector by using BsaI, and then carrying out gel electrophoresis to recover an enzyme digestion product with the size of about 15 kb;

s13, inserting double-stranded DNA formed by annealing forward and reverse primers of sgRNA1 and sgRNA2 into a linearized vector of an enzyme digestion product in S12 to obtain recombinant vectors pKSE401-sgRNA1 and pKSE401-sgRNA 2;

s2, constructing a BSMV-mediated sgRNA recombinant vector;

s21, performing single enzyme digestion on the BSMV-gamma, BSMV-gamma 1 and BSMV-gamma 2 virus vectors by using ApaI, and recovering enzyme digestion products by gel electrophoresis;

s22, using Q5 high-fidelity DNA polymerase and sgRNA Ti vector as a template, amplifying sgRNA1 and sgRNA2 fragment sequences by PCR, and recovering amplification products by gel electrophoresis;

s23, connecting the linearized vector in S21 and the sgRNA fragment in S22 by a ligation method of LIC to form a recombinant vector BSMV-gamma: sgRNA2, BSMV- γ 1: sgRNA1, BSMV- γ 2: sgRNA1 and BSMV- γ 1: sgRNA2, BSMV- γ 2: sgRNA 2;

s3, constructing a BSMV-mediated split-Cas9 recombinant vector;

s31, performing single enzyme digestion on the BSMV-gamma 2 viral vector by using ApaI, and recovering an enzyme digestion product by gel electrophoresis;

s32, splitting the spCas9 from the 714(S) th amino acid, and using Q5 high-fidelity DNA polymerase and pKSE401 vector as a template to respectively amplify a Cas9N end and a Cas9C end to obtain a split-Cas9 fragment;

s33, connecting the linearized vector of the enzyme digestion product in S31 with the split-Cas9 fragment in S32 by a LIC connection method to form a recombinant vector BSMV-gamma 2: cas9N, BSMV- γ 2: cas 9C.

Further, the forward and reverse primers of sgRNA1 and sgRNA2 in S13 are:

sgRNA1 forward primer sequence: 5'-ATTGTTGGTAGTAGCGACTCCATG-3'

sgRNA1 reverse primer sequence: 5'-AAACCATGGAGTCGCTACTACCAA-3'

sgRNA2 forward primer sequence: 5'-ATTGGAGGCAAGAGATGTCCTAGG-3'

sgRNA2 reverse primer sequence: 5'-AAACCCTAGGACATCTCTTGCCTC-3'

Further, the Cas9N end PCR primers in S32 are:

forward primer sequence:

5’-AAGGAAGTTTAAATGGATTACAAGGACCACGA-3’

reverse primer sequence:

5’-CGGGCCAGCCACCGCCACCAGTGCTCACCTGAGCCTT-3’。

further, the Cas9C end PCR primers in S32 are:

forward primer sequence:

5’-AAGGAAGTTTAAATGGGCCAGGGGGACTCGCT-3’

reverse primer sequence:

5’-CGGGCCAGCCACCGCCACCAGTTCACTTCTTCTTCTTCGCC-3’。

the invention also discloses an application of the BSMV viral vector-mediated CRISPR/Cas9 recombinant vector.

The invention also discloses application of the BSMV viral vector-mediated CRISPR/Cas9 recombinant vector to delivering tobacco leaves of Bunshi which overexpress SpCas 9.

The invention also discloses application of the BSMV viral vector-mediated CRISPR/Cas9 recombinant vector in non-transgenic editing of plants.

The invention can obtain the following technical effects:

1) the invention utilizes Barley Stripe Mosaic Virus (BSMV) to recombine and construct a BSMV vector system containing three different single-stranded RNAs of alpha, beta and gamma or recombine and construct a BSMV vector system containing four different single-stranded RNAs of alpha, beta, gamma 1 and gamma 2. Firstly, verifying the feasibility of sgRNA delivery of a three-component BSMV and four-component BSMV system in tobacco (KQ334) exceeding Cas9 to achieve a gene editing result; and then, the feasibility of co-delivering sgRNA + Cas9 by a four-component BSMV system is verified, so that a non-transgenic editing result is achieved.

2) The recombinant vector can generate systematic editing results in plant target genes, establishes an effective method for researching plant gene functions, and provides a new thought for new plant gene editing.

3) Compared with a lengthy agrobacterium-mediated genetic transformation process, the system has obvious advantages, has important significance for the application research of the RNA virus-mediated CRISPR/Cas9 system in plant gene editing, and lays a foundation for further utilizing a virus vector to infect reproductive tissues so as to directly obtain a target of a non-transgenic editing seed.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a schematic diagram of pKSE401 vector;

fig. 2 shows sgRNA1 and sgRNA2 of selected tobacco PDS genes and preliminary validation of their editing activity;

FIG. 3 is a schematic diagram of the main structure of a BSMV vector, wherein A is a three-component BSMV system and B is a four-component BSMV system;

FIG. 4 is a schematic diagram of the principle of LIC (ligation independent cloning);

fig. 5 is a graph of the results of a PCR/RE experiment with three-component BSMV delivery of sgRNA 2;

fig. 6 is a graph of the results of a PCR/RE experiment with four-component BSMV delivery of sgRNA 2;

FIG. 7 is a graph showing the results of PCR detection of large fragment deletion by four-component BSMV delivery of double sgRNAs (sgRNAs 1+ sgRNAs 2);

FIG. 8 is a simplified schematic illustration of split-Cas 9;

fig. 9 is a graph of the results of a PCR/RE experiment with four-component BSMV co-delivery of sgRNA2+ Cas 9.

Detailed Description

The present invention will be described in further detail with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The invention provides a new idea for plant gene editing. The research utilizes the characteristics of strong infectivity and mobility of plant viruses and the like, and can systematically infect various plants. Compared with the traditional binary vector-mediated CRISPR/Cas9 technology, the virus-mediated plant gene editing has greatly improved editing efficiency due to continuous virus replication, and has strong mobility and can generate effective mutation on the whole plant. Compared with the most common transgenic technology at present, the invention provides a novel method for non-transgenic editing, avoids the disputes caused by transgenic problems, is relatively safe, greatly reduces risks, and provides possibility for the subsequent application in the aspect of crops which are difficult to transform due to the broad spectrum of the invention. The research has important significance for the application research of the RNA virus mediated CRISPR/Cas9 system in plant gene editing, and lays a foundation for further infecting reproductive tissues by using a virus vector and further obtaining the target of non-transgenic editing seeds. The four-component BSMV vector system mentioned in the research has wider application prospect to a gene editing system with smaller size due to the limited loading capacity, and provides an important basis for the following non-transgenic editing research mediated by a virus vector.

The test methods used in the following examples are all conventional methods unless otherwise specified.

The invention relates to vectors or plasmids, such as BSMV-alpha,BSMV-β、BSMV-γ、pMD^TMThe 19-T Vector and the like are all known vectors or plasmids, BSMV-alpha, BSMV-beta and BSMV-gamma referred to in the following examples are given as gifts from the professor Lidami David of China university of agriculture, and BSMV-gamma 1 and BSMV-gamma 2 can be constructed by themselves according to the existing literature.

The plant material, namely the transgenic SpCas9-OE Benzilian tobacco, is presented by professor Liuyule of Qinghua university, and other plant materials involved in the embodiment can be obtained from a commercial way without special instructions.

Example 1 construction of sgRNA Ti vectors

According to the design principle of spCas9 PAM sequence (NGG) and optimal length of 20nt, two sgRNAs are designed and respectively connected into pKSE401 vector.

Design of sgRNA:

finding the CDS sequence of the tobacco PDS gene on NCBI;

finding the genome sequence of the tobacco PDS in a tobacco genome website by using the sequence, and obtaining exon sequence information of the tobacco PDS gene;

designing an sgRNA target sequence of the NbPDS gene by using on-line software CRISPR-P software according to the obtained exon sequence information of the tobacco PDS gene;

two sgrnas with suitable cleavage sites (sgRNA1 and sgRNA2) were obtained by analysis.

The construction of the sgRNA Ti vector specifically comprises the following steps:

(1) target site design primer selection:

sgRNA1 forward primer sequence: 5'-ATTGTTGGTAGTAGCGACTCCATG-3'

sgRNA1 reverse primer sequence: 5'-AAACCATGGAGTCGCTACTACCAA-3'

sgRNA2 forward primer sequence: 5'-ATTGGAGGCAAGAGATGTCCTAGG-3'

sgRNA2 reverse primer sequence: 5'-AAACCCTAGGACATCTCTTGCCTC-3'

(2) Using BsaI to singly digest pKSE401 vector, and carrying out gel electrophoresis to recover a digest product with the size of about 15 kb;

(3) annealing the forward and reverse primers of the sgRNA1 and the sgRNA2 in the step (1) to form double-stranded DNA, and specifically performing the following steps: dissolving the two synthesized primers into 100uM by using deionized water, uniformly mixing 5ul of the two primers, carrying out water bath at 80 ℃ for 15min, taking out, and naturally cooling to room temperature to complete the process; inserting the double-stranded DNA into a linearized vector of the enzyme digestion product in the step (2) to obtain recombinant vectors pKSE401-sgRNA1 and pKSE401-sgRNA 2;

(4) transforming the recombinant vectors pKSE401-sgRNA1 and pKSE401-sgRNA2 into escherichia coli competent cells, randomly selecting monoclonals to culture in an LB liquid culture medium at 37 ℃ overnight after the plate grows out the monoclonals, identifying the recombinant plasmids by a bacterial liquid PCR combined sequencing method, and preparing a large amount of the required recombinant plasmids after the identification is correct.

BasI single enzyme digestion system: mu.L of CutSmart buffer, 0.2. mu.L of BsaI, 5. mu.L of plasmid, 3.8. mu.L of ddH₂O; reaction conditions are as follows: the enzyme was cleaved at 37 ℃ for 1 h.

T4 ligase ligation reaction system: 3. mu.L of linearized vector, 5. mu.L of double-stranded DNA, 1. mu.L of 10 XLigase buffer, 1. mu. L T4 DNA Ligase; reaction conditions are as follows: connecting for 2-3 h at 16 ℃.

FIG. 1 shows a schematic diagram of pKSE401 vector; fig. 2(a) shows selected sgrnas 1 and 2; fig. 2(B) shows sgRNA1 editing efficiency results, and fig. 2(C) shows sgRNA2 editing efficiency results. The results indicate that both sgRNA1 and sgRNA2 selected can result in editing of the target gene PDS.

Example 2: construction of four-component BSMV recombinant vectors:

the three-component BSMV systems (BSMV-alpha, BSMV-beta and BSMV-gamma) are from the professor Rivere great Living of China university of agriculture; with reference to 2017(Mario Houde 2017), a four-component BSMV system (BSMV-alpha, BSMV-beta, BSMV-gamma 1 and BSMV-gamma 2) is constructed, and can be used for CRISPR/Cas9 reagent delivery.

Fig. 3(a) shows a three-component BSMV system vector schematic, and fig. 3(B) shows a four-component BSMV system vector schematic.

Example 3: ligation Independent Cloning (LIC) method

FIG. 4 shows the principle diagram of LIC reaction, which is a ligase-independent cloning method with high cloning efficiency and short reaction time, and the LIC method provides conditions for high-throughput cloning operation. LIC utilizes the exo-activity of T4 polymerase 3 '-5' end, and is a designed sequence that can be exo-ligated to generate a longer cohesive end (typically about 15 bp) of a specific length and specific sequence. When the cohesive ends generated by the vector and the PCR fragment were treated to complementarily match each other, the complementary pair of 15bp in length was able to maintain the circular structure of the recombinant plasmid in the absence of ligase by denaturation and annealing. This unligated recombinant plasmid is transformed into E.coli, and the complete circular recombinant plasmid is generated using the replication system of E.coli. Wherein the reaction procedure is as follows:

firstly, the LIC vector is linearized, the PCR product of the target fragment is recovered and purified, and then a reaction solution is prepared according to the following system:

TABLE 1 formulation of linearized viral vectors

Reagent composition	Dosage of
		Linearized viral vectors	100ng
NEBuffer 2.1	2μL
		T4 DNA polymerase	0.5μL
100mM dTTP	1.5μL
		Supplement with water	20μL

TABLE 2 preparation of recovered fragments

Reagent composition	Dosage of
		Recovering the fragments	100ng
NEBuffer 2.1	2μL
		T4 DNA polymerase	0.5μL
100mM dATP	1.5μL
		Supplement with water	20μL

Reaction conditions are as follows: reacting at 22 deg.C for 30min, reacting at 75 deg.C for 20min to inactivate enzyme, mixing 10ng treated carrier and 100ng treated recovered fragment, heating to 66 deg.C, reacting for 2min, standing at room temperature for 10min, and transforming Escherichia coli competent cells.

Example 4: BSMV-mediated gene editing of sgRNA recombinant vectors

And constructing a BSMV mediated sgRNA recombinant vector by using the constructed pKSE401-sgRNA1 and pKSE401-sgRNA2 as templates through a LIC (laser induced ligation) connection method.

The construction method comprises the following steps:

(1) sgRNA viral vector fragment primer selection, as shown in table 3:

table 3sgRNA viral vector fragment primers

(2) Using ApaI to singly cut BSMV-gamma, BSMV-gamma 1 and BSMV-gamma 2 virus vectors, and recovering the cut products by gel electrophoresis;

(3) using Q5 high-fidelity DNA polymerase, using the recombinant vector in the above example 1 as a template, selecting appropriate primers in the step (1) to amplify each sgRNA fragment sequence by PCR, and recovering the enzyme digestion product by gel electrophoresis;

(4) and (3) connecting the linearized vector of the enzyme digestion product in the step (2) and the sgRNA fragment in the step (3) by a LIC (restriction enzyme cleavage nucleic acid) connection method to form a recombinant vector BSMV-gamma: sgRNA2, BSMV- γ 1: sgRNA1, BSMV- γ 2: sgRNA1 and BSMV- γ 1: sgRNA2, BSMV- γ 2: sgRNA 2;

(5) transforming the recombinant vector into escherichia coli competent cells, randomly selecting monoclonals to culture in an LB liquid culture medium at 37 ℃ overnight after the monoclonals grow out from the plate, identifying the recombinant plasmids by a bacterial liquid PCR combined sequencing method, and preparing a large amount of required recombinant plasmids after the recombinant plasmids are identified correctly.

And (3) PCR reaction system: 4 μ L of 5 XQ 5 Reaction Buffer, 1.6 μ L of 2.5mM dNTP, 0.8 μ L of 10 μ M Forward Primer, 0.8 μ L of 10 μ M Reverse Primer, 1 μ L of recombinant vector template, 0.2 μ L Q5 High-Fidelity DNA Polymerase, 11.6 μ L of ddH₂O; reaction procedure: denaturation at 98 ℃ for 10s, annealing at 57 ℃ for 10s, extension at 72 ℃ for 30s, and circulation for 35 times.

ApaI single enzyme digestion system: mu.L of CutSmart buffer, 0.2. mu.L of ApaI, 5. mu.L of viral vector plasmid, 3.8. mu.L of ddH₂O; reaction conditions are as follows: the enzyme was cleaved at 37 ℃ for 1 h.

(II) delivering the BSMV-mediated sgRNA recombinant vector to the overexpression SpCas9-OE Nihon bungii, and specifically operating steps of:

(1) selecting Agrobacterium clones containing BSMV mediated sgRNA recombinant vectors, inoculating to liquid YEB culture medium containing 25ug/ml rifampicin and 50ug/ml kana, and culturing at 28 deg.C and 200r/min under shaking for 24 h;

inoculating 1ml (at a ratio of 1: 50) of the above Agrobacterium strain solution into a new liquid YEB culture medium containing 25ug/ml rifampicin and 50ug/ml kanamycin, and performing shake culture at 28 deg.C and 200r/min for 8-10 hr;

centrifuging the obtained bacterial liquid at 20 deg.C for 10min at 5000r/min to collect thallus, then suspending the thallus in MMA-10mmol/L,10mmol/LMES,100umol/L AS to obtain thallus suspension, and adjusting OD₆₀₀＝0.3；

(2) Respectively taking the BSMV-alpha, BSMV-beta, BSMV-gamma-sgRNA 2 or BSMV-alpha, BSMV-beta, BSMV-gamma 2 and BSMV-gamma 1 with equal volumes: sgRNA2 or BSMV- α, BSMV- β, BSMV- γ 1, BSMV- γ 2: sgRNA2 or BSMV- α, BSMV- β, BSMV- γ 1: sgRNA1, BSMV- γ 2: sgRNA2 or BSMV- α, BSMV- β, BSMV- γ 1: sgRNA2, BSMV- γ 2: uniformly mixing sgRNA1 to form a BSMV three-component system or four-component system, and culturing in the dark at 28 ℃ for 3-4 hours;

(3) selecting 6-8 leaf stage over-expressed SpCas9-OE Nicotiana benthica, lightly touching the back surface of the 6-8 leaf stage over-expressed SpCas9-OE Nicotiana benthica with a needle to cause a micro wound, then respectively sucking the mixed bacterial liquid in the step (2) by using an injector without the needle to slowly inject the mixed bacterial liquid into the tobacco leaves through the wound, and selecting 3-4 holes for each leaf to inject until the whole leaf is soaked by the bacterial liquid, and stopping injecting;

(4) after the injection is finished, spraying a small amount of clear water on infected leaves, carrying out dark treatment for 24h, and carrying out normal light cycle maintenance for 7 d.

(III) feasibility test

(1) Extracting the genome DNA of infected leaf and systemic leaf of experimental treatment Ben's tobacco

(2) PCR and sequencing detection of Gene editing types

(I) single sgRNA delivery:

using the extracted tobacco leaf DNA as a template, amplifying PCR fragments of corresponding target sites of NbPDS3 gene by using high-fidelity enzyme, after recovering PCR products, respectively using AvrIII restriction enzyme to enzyme-cut the PCR amplified fragments of the corresponding target sites, after enzyme cutting, detecting the enzyme cutting result by using 2% agarose gel electrophoresis, recovering the cut gel of the target fragment, and connecting the recovered cut gel into a sequencing vector pMD^TM19-T Vector, wherein,

PCR primers for sgRNA2 were:

5’-GCATGGTACTGTGCCGATCA-3’

5’-GCAACCCAGTCTCGTACCAA-3’

(II) double sgRNA delivery:

using extracted tobacco leaf DNA as template, using high-fidelity enzyme to amplify PCR fragment of NbPDS3 gene including two target sites, using wild type as contrast, using 2% agarose gel electrophoresis to detect PCR result, analyzing gel picture to determine whether fragment deletion mutation exists, cutting and recovering target fragment, connecting it into sequencing vector pMD^TM19-T Vector for sequencing analysis, wherein the PCR primers for detecting deletion are as follows:

5’-AGGTTCACAAGTGGGACAATC-3’

5’-GCAACCCAGTCTCGTACCAA-3’

and (3) PCR reaction system: 4 μ L of 5 XQ 5 Reaction Buffer, 1.6 μ L of 2.5mM dNTP, 0.8 μ L of 10 μ M Forward Primer, 0.8 μ L of 10 μ M Reverse Primer, 1 μ L of recombinant vector template, 0.2 μ L Q5 High-Fidelity DNA Polymerase, 11.6 μ L of ddH₂O; reaction procedure: denaturation at 98 ℃ for 10s, annealing at 57 ℃ for 10s, extension at 72 ℃ for 30s, and circulation for 35 times.

NcoI single enzyme system: mu.L of CutSmart buffer, 0.2. mu.L of NcoI, 5. mu.L of LPCR product, 3.8. mu.L of ddH₂O; reaction conditions are as follows: the enzyme was cleaved at 37 ℃ for 1 h.

Fig. 5 shows sgRNA2 mediated editing of tobacco PDS genes by the three-component BSMV system delivery; the results show that the three-component BSMV system can systematically infect tobacco plants, and gene editing results can be detected in both injection leaves and systemic leaves. Fig. 6 shows sgRNA2 mediated editing of tobacco PDS genes by four-component BSMV systemic delivery; the results show that the four-component BSMV system can systematically infect tobacco plants, and gene editing results can be detected in both injection leaves and systemic leaves. Fig. 7 shows four-component BSMV systemic delivery of sgRNA1 and sgRNA2 mediated editing of tobacco PDS genes; the results indicate that a four-component system can deliver two sgrnas simultaneously and can result in large fragment deletions.

Example 5: four-component BSMV Co-delivery sgRNA + Cas 9-mediated non-transgenic editing

A BSMV-mediated split-Cas9 recombinant vector is constructed by a LIC ligation method by taking pKSE401 as a template.

The construction method comprises the following steps:

(1) using ApaI to singly cut pCaBs-gamma 2 viral vectors, and carrying out gel electrophoresis recovery;

(2) referring to (Jorrit Boekel 2015), the spCas9 was split from its 714(S) th amino acid, using Q5 high fidelity DNA polymerase with pKSE401 vector as template, to amplify Cas9N and Cas9C ends, respectively; wherein, the PCR primer at the Cas9N end is as follows:

a forward primer:

5’-AAGGAAGTTTAAATGGATTACAAGGACCACGA-3’

reverse primer:

5’-CGGGCCAGCCACCGCCACCAGTGCTCACCTGAGCCTT-3’

the Cas9C end PCR primers were:

a forward primer:

5’-AAGGAAGTTTAAATGGGCCAGGGGGACTCGCT-3’

reverse primer:

5’-CGGGCCAGCCACCGCCACCAGTTCACTTCTTCTTCTTCGCC-3’

(3) and (3) connecting the linearized vector in the step (I) and the split-Cas9 fragment in the step (II) by a connecting method of LIC to form a recombinant vector pCaBs-gamma 2: cas9N, pCaBs- γ 2: cas 9C;

(4) the recombinant vector pCaBs-gamma 2: cas9N, pCaBs- γ 2: cas9C transforms escherichia coli competent cells, after a plate grows out a single clone, randomly selecting the single clone to be cultured overnight in LB liquid medium at 37 ℃, identifying recombinant plasmids by using a method of bacteria liquid PCR combined with sequencing, preparing a large amount of required recombinant plasmids after correct identification, then transforming agrobacterium, identifying positive clones, and storing by streaking.

(II) delivering the BSMV-mediated split-Cas9 recombinant vector to wild type Nicotiana benthamiana, and specifically operating steps of:

(1) selecting Agrobacterium clones containing BSMV mediated split-Cas9 recombinant vector, inoculating into liquid YEB culture medium containing 25ug/ml rifampicin and 50ug/ml kana, and culturing at 28 deg.C and 200r/min under shaking for 24 h;

inoculating 1ml (at a ratio of 1: 50) of the above Agrobacterium strain solution into a new liquid YEB culture medium containing 25ug/ml rifampicin and 50ug/ml kanamycin, and performing shake culture at 28 deg.C and 200r/min for 8-10 hr;

centrifuging the obtained bacterial liquid at 20 deg.C for 10min at 5000r/min to collect thallus, then suspending the thallus in MMA-10mmol/L,10mmol/LMES,100umol/L AS to obtain thallus suspension, and adjusting OD₆₀₀＝0.3；

(2) Respectively taking the BSMV-alpha, BSMV-beta and BSMV-gamma 1 with equal volumes: sgRNA2, BSMV- γ 2: cas9N or BSMV- α, BSMV- β, BSMV- γ 1: sgRNA2, BSMV- γ 2: cas9C is uniformly mixed to form a BSMV eight-component system, and is cultured in the dark at 28 ℃ for 3-4 hours;

(3) selecting wild type Nicotiana benthamiana in 6-8 leaf stages, lightly touching the back of the wild type Nicotiana benthamiana with a needle to cause a micro wound, then respectively sucking the mixed bacterial liquid in the step (2) by using an injector without the needle to slowly inject the mixed bacterial liquid into the tobacco leaves through the wound, and selecting 3-4 holes for each leaf to inject until the whole leaf is soaked by the bacterial liquid, and stopping injecting;

(4) after injection, spraying a small amount of clear water on infected leaves, carrying out dark treatment for 24h, and carrying out normal light cycle maintenance for 7 d;

(5) extracting RNA, performing reverse transcription to obtain cDNA, and performing semi-quantitative detection on virus accumulation condition;

(6) extracting the genome DNA of the tobacco leaf by using a CTAB method, and grinding a sample by using liquid nitrogen;

(7) using the extracted tobacco leaf DNA as a template, amplifying PCR fragments of corresponding target sites of NbPDS3 gene by using high-fidelity enzyme, after recovering PCR products, using AvrII restriction endonuclease to cut the PCR amplified fragments of the corresponding target sites, after cutting, using 2% agarose gel electrophoresis to detect the cut enzyme results, recovering the cut gel of the target fragments, and connecting the cut gel into a sequencing vector pMD^TM19-T Vector for sequencing analysis, wherein PCR primers for sgRNA2 were:

a forward primer: 5'-GCATGGTACTGTGCCGATCA-3'

Reverse primer: 5'-GCAACCCAGTCTCGTACCAA-3'

And (3) PCR reaction system: 4 μ L of 5 XQ 5 Reaction Buffer, 1.6 μ L of 2.5mMdNTP, 0.8. mu.L 10. mu.M Forward Primer, 0.8. mu.L 10. mu.M Reverse Primer, 1. mu.L recombinant vector template, 0.2. mu. L Q5 High-Fidelity DNA Polymerase, 11.6. mu.L ddH₂O; reaction procedure: denaturation at 98 ℃ for 10s, annealing at 57 ℃ for 10s, extension at 72 ℃ for 30s, and circulation for 35 times.

NcoI single enzyme system: mu.L of CutSmart buffer, 0.2. mu.L of NcoI, 5. mu.L of LPCR product, 3.8. mu.L of ddH₂O; reaction conditions are as follows: the enzyme was cleaved at 37 ℃ for 1 h.

FIG. 8 shows a schematic diagram of Split-Cas 9; fig. 9 shows four-component BSMV co-delivery sgRNA + Cas 9-mediated gene editing. The results show that the four-component BSMV system can co-deliver sgRNA + Cas9 resulting in gene editing results.

The above results indicate that the BSMV-mediated sgRNA recombinant vector is able to generate an editing event in native tobacco overexpressing spCas9, and in systemic leaves, the editing event was also detected (see fig. 5, 6). For the double-site editing, the PCR result shows a large 834bp band (as shown in FIG. 7), which indicates that the recombinant vector can mutate two target sites simultaneously and has high editing efficiency. For non-transgenic editing, the results show that the BSMV four-component system can achieve co-delivery of sgRNA + Cas9, and the PCR/RE experimental results show the generation of editing events (see fig. 9).

In conclusion, the BSMV-mediated sgRNA delivery and sgRNA + Cas9 co-delivery system can be applied to CRISPR/Cas9 for gene editing of plants.

While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.