Establishment and application of plant virus mediated ABE base editing system in cotton

Abstract

The invention relates to the technical field of gene engineering, in particular to establishment and application of a plant virus mediated ABE base editing system in cotton. According to the invention, CLCrV is used as a delivery medium, nCas9-TadA7.10-OE cotton is used as a transformation receptor, and an ABE base editing system based on CLCrV mediation is constructed. A gene expression quantity result shows that nCas9 is stably genetically expressed in different transgenic lines. Different constructed CLCrV-ABEs virus vectors are inoculated to nC as9-TadA7.10-OE cotton, mutation detection is carried out, a deep sequencing result shows that CLCrV-AtU6-26: sgGhPEBP-ABE can effectively cause conversion from an A basic group to a G basic group of a GhPEBP gene target site, and the editing efficiency is 6.18%-29.26%. Meanwhile, off-target detection is carried out in a plant subjected to base editing of GhPEBP, and the result shows that no off-target phenomenon is detected on the predicted potential off-target site, so that the CLCrV-mediated ABE base editing system has certain gene editing specificity. The establishment of the plant virus-mediated ABE base editing system in cotton provides a favorable technical tool for screening efficient sgRNAs in cotton and designing and breeding cotton molecules.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to the establishment and application of a plant virus-mediated ABE base editing system in cotton. Background Art

[0002] Genetically variable mutants are the basis for analyzing crop gene functions and the formation of important agronomic traits. In the field of crop breeding research, CRISPR / Cas genome editing technology can overcome the shortcomings of random mutations caused by physical and chemical mutagenesis and T-DNA insertion, and achieve targeted editing at specific sites in the genome. Cas9, Cas12a (Cpf1), base editing (ABE and CBE) and guide editing systems have been successfully established in a variety of plants and crops. However, for most plants and crops, genetic transformation is still the main bottleneck for using these gene editing technologies to achieve target gene editing and create gain-of-function mutants, and cotton is no exception.

[0003] Plant virus vectors have the characteristics of autonomous replication and movement in host cells, and are ideal transient transformation vectors. Plant viruses are mainly divided into two categories according to their genetic material: RNA and DNA viruses. Many studies have shown that some plant viruses can be modified and assembled on plant expression vectors to achieve gene silencing and overexpression by Agrobacterium infection, and even gene editing, which is called virus-induced genome editing (VIGE). Cotton leaf crumple virus (CLCrV) is a DNA virus that can systematically and persistently infect cotton. Replacing its coat protein with a short gene silencing fragment can effectively knock down the expression of cotton endogenous genes and produce a gene silencing phenotype. CLCrV can also be used in cotton gene editing research. Using a truncated Arabidopsis or cotton endogenous U6 promoter to drive sgRNA with a target sequence to replace the coat protein of CLCrV-A can efficiently achieve targeted editing of endogenous genes in Arabidopsis and cotton overexpressing Cas9 (Cas9-OE), and obvious mutant phenotypes can also be observed for some visual marker genes. However, the CLCrV-mediated base editing system in cotton has not yet been established, and whether the system can achieve targeted base editing of target genes still needs further testing. Previously, studies have reported that the use of potato virus X (PVX) to deliver a complete CBE base editor in wild-type tobacco can achieve the conversion of C bases to T bases at the target site of the NbTOM1 gene. Liu et al. designed sgRNAs for the Arabidopsis AtPDS3, AtCLA1, and AtCESA3 genes and assembled them on tobacco rattle virus (TRV). Arabidopsis inoculated with overexpressed CBE base editors can also achieve the conversion of target gene C bases to T bases, and can achieve heritable gene editing. The development of these virus-mediated base editing tools provides a simple and fast technical tool for screening efficient sgRNAs and studying plant gene functions.

[0004] Unlike PVX, CLCrV does not have the characteristics of delivering complete CRISPR / Cas components in cotton. Using nCas9-TadA7.10-OE cotton transgenic material as VIGE receptor provides a good VIGE receptor material for establishing the CLCrV-mediated ABE base editing system. By constructing a CLCrV-mediated ABE base editing system, we hope to achieve base editing in cotton in a simple and fast way, and provide a favorable technical tool for cotton gene function research. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a plant virus-mediated ABE base editing system in cotton for the above-mentioned deficiencies of the prior art, and to use the plant virus CLCrV as a delivery medium and nCas9-TadA7.10-OE cotton as a transformation receptor to construct an ABE base editing system based on the plant virus CLCrV. The establishment of the plant virus-mediated ABE base editing system in cotton provides a favorable technical tool for screening efficient sgRNAs in cotton and molecular design breeding of cotton.

[0006] In order to solve the above technical problems, the present invention provides the establishment of a plant virus-mediated ABE base editing system in cotton, wherein the ABE base editing system uses CLCrV as a delivery medium and nCas9-TadA7.10-OE cotton as a transformation receptor to construct an ABE base editing system mediated by CLCrV.

[0007] According to the establishment of the plant virus-mediated ABE base editing system in cotton provided by the present invention, the ABE base editing system mediated by the plant virus CLCrV in cotton can achieve targeted mutation of the target gene (GhPEBP).

[0008] According to the establishment of the plant virus-mediated ABE base editing system in cotton provided by the present invention, the editing efficiency of the ABE base editing system mediated by the plant virus CLCrV in cotton for the GhPEBP gene is 6.18% to 29.26%.

[0009] According to the establishment of the plant virus-mediated ABE base editing system in cotton provided by the present invention, the ABE base editing system mediated by the plant virus CLCrV in cotton has certain gene editing specificity.

[0010] The present invention also provides an application of the establishment of the above-mentioned plant virus-mediated ABE base editing system in cotton, wherein the plant virus CLCrV-mediated ABE base editing system in cotton can be used for screening efficient sgRNAs in cotton and cotton molecular design breeding.

[0011] Compared with the prior art, the present invention has the following advantages:

[0012] The present invention uses plant virus CLCrV as a delivery medium and nCas9-TadA7.10-OE cotton as a transformation receptor to construct an ABE base editing system mediated by plant virus CLCrV in cotton. Compared with the method of transforming wild-type cotton protoplasts by constructing a complete CRISPR / nCas9-TadA7.10 base editor or transforming wild-type cotton cotyledons in the form of Agrobacterium, this system can simply and quickly realize target gene base editing, greatly improve the efficiency of gene mutation, and provide a favorable technical system for quickly screening sgRNAs that can efficiently edit target genes in cotton. At the same time, the system detects potential off-target effects in plants where base editing of target genes occurs. Through deep sequencing, no off-target phenomenon is detected at the predicted potential off-target sites, indicating that the ABE base editing system mediated by plant virus CLCrV in cotton has certain gene editing specificity.

[0013] In summary, the plant virus-mediated ABE base editing system in cotton provides a favorable technical tool for screening efficient sgRNAs in cotton and molecular design breeding of cotton. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is qPCR to detect nCas9 expression;

[0015] Figure 2 It is the restriction enzyme identification of CLCrV-ABEs recombinant plasmid;

[0016] Figure 3 It is PCR amplification of 4 target genes covering the mutation sites;

[0017] Figure 4 It is the GhPEBP mutation genotype detection;

[0018] Figure 5 is the PCR amplification of ProAtU6-26::sgRNA and the detection of viral accumulation;

[0019] Figure 6 It is a sequence comparison analysis of 3 potential off-target sites. DETAILED DESCRIPTION

[0020] Example 1

[0021] ①Material selection:

[0022] The raw materials of this embodiment include AtU6-26::sgRNA vector, cotton leaf crinkle virus (CLCrV) vectors CLCrV-A and CLCrV-B, and nCas9-TadA7.10-OE cotton material.

[0023] In order to establish a CLCrV-mediated ABE base editing system in cotton, cotton leaf crinkle virus (CLCrV) was used as a delivery vehicle and nCas9-TadA7.10-OE cotton was used as a transformation receptor to construct an ABE base editing system mediated by the plant virus CLCrV in cotton.

[0024] ②nCas9 expression detection:

[0025] Wild-type and different strains of nCas9-TadA7.10-OE cotton seeds were sown in nutrient soil with a ratio of vermiculite to black soil of 3:1, and cultured at a temperature of 28°C and a light intensity of 16 h light culture / 8 h dark culture.

[0026] When the two cotyledons of cotton were fully expanded, some plants of the same strain were selected, and the wild-type and nCas9-TadA7.10-OE cotton cotyledon RNA was extracted according to the operation steps of the Plant Polysaccharide and Polyphenol RNA Extraction Kit, and cDNA was synthesized after confirming the integrity of the RNA. Using cDNA as a template, qPCR amplified the nCas9 gene, and the cotton GhUBQ7 gene was used as an internal reference gene. The primer design is shown in Table 1.

[0027] The qPCR reaction system was as follows: 1 μL cDNA, 0.5 μL upstream and downstream primers nCas9F / nCas9R, 10 μL qPCRMix, 8 μL Nuclease-free H2O; the reaction program was as follows: 95°C for 30 s; 95°C for 5 s, 60°C for 30 s, 40 cycles; 3 sets of technical replicates were set for each sample, and finally the results were analyzed according to 2 -△△Ct The algorithm calculates the relative expression of the nCas9 gene.

[0028] Table 1 Primer sequences

[0029]

[0030]

[0031]

[0032] ③CLCrV-ABEs vector construction:

[0033] Based on the GhCLA1, GhPDS, and GhPEBP genomic sequences actually cloned on the nCas9-TadA7.10-OE cotton A / D subgroup, sgRNAs that could simultaneously edit the cotton GhCLA1-A / D, GhPDS-A / D, and GhPEBP-A / D genes were designed in the exon region.

[0034] The target sequence was synthesized into sgRNA by annealing and assembled on the At U6-26::sgRNA vector linearized by Bbs I. The correctness of AtU6-26::sgGhCLA1-ABE1, AtU6-26::sgGhCLA1-ABE2, AtU6-26::sgGhPDS-ABE and AtU6-26::sgGhPEB P-ABE plasmids was verified by sequencing.

[0035] The above sequenced plasmids were digested with SpeⅠ and PacⅠ, and the target gene fragments were recovered and purified and recombined with the linearized CLCrV-A vector to generate CLCrV-AtU6-26::sgGhCLA1-ABE1, CLCrV-AtU6-26::sgGhCLA1-ABE2, CLCrV-AtU6-26::sgGhPDS-ABE and CLCrV-AtU6-26::sgGhPEBP-ABE recombinant plasmids. Finally, the above CLCrV-ABEs final vectors were transformed into Agrobacterium competent GV3101 for use.

[0036] ④CLCrV-ABEs inoculation and transformation of nCas9-TadA7.10-OE cotton:

[0037] The Agrobacterium tumefaciens of different CLCrV-ABEs and CLCrV-B were streaked and single clones were picked and inoculated into 2 mL of 50 μg mL -1 Kan and 50 μg mL -1 Rif was activated in LB medium and cultured at 28°C and 200 r / min for 24 h. 60-100 μL of activated bacterial solution was transferred to 10 mL of LB medium containing the above antibiotics and 200 μmol acetosyringone, and the bacteria were shaken at 28°C and 200 r / min until the OD600 of the bacterial solution was about 1.5-1.8, and the bacteria were collected by centrifugation at 3000×g for 15 min.

[0038] The different CLCrV-ABEs Agrobacterium cultures were resuspended in 10 mmol·L transformation solution. -1 MgCl2, 10mmol·L -1 MES and 200 μmol·L -1 Acetosyringone was added and the final OD600 concentration was adjusted to about 1.0. Finally, CLCrV-B was mixed with different CLCrV-ABEs Agrobacterium resuspensions at a ratio of 1:1. After standing for 3 to 4 hours in the dark, the lower epidermis of the cotton cotyledons was pierced with a 1 mL sterile syringe needle, and the different CLCrV-ABEs and CLCrV-B mixtures were inoculated into the cotton cotyledons with a syringe.

[0039] ⑤Target gene mutation detection and editing efficiency analysis:

[0040] Ten days after inoculation of the viral vector, genomic DNA was extracted from the cotyledons of cotton inoculated with different CLCrV-ABEs. Using the genomic DNA of the wild type and each inoculated individual as templates, Hi-TOM high-throughput sequencing primers were designed, as shown in Table 1. PCR amplification covering the mutation sites of GhCLA1, GhPDS and GhPEBP genes was performed. The PCR reaction system was 4 μL 5×PhusionBuffer, 1.6 μL 2.5mmol / L dNTP, 1 μL of upstream and downstream primers (high-throughput sequencing adapters were added to the 5' end of the target gene-specific amplification primers) F / R, 1 μL DNA, 0.2 μL Phusion high-fidelity enzyme, and ddH2O was added to 20 μL. The reaction program was 98℃30s; 98℃10s, 60℃30s, 72℃10s (35 cycles); 72℃ final extension for 7min. After the reaction, 3 μL of the target gene PCR amplification product was aspirated for agarose gel electrophoresis detection, and the PCR product with the correct target fragment size was selected for Hi-TOM high-throughput sequencing. After the high-throughput sequencing, the mutation of the target gene in the cotton A / D subgroup was analyzed, and the gene editing efficiency was counted. At the same time, plants with higher gene editing efficiency were selected for Sanger sequencing to further clarify the mutant genotype of the target gene.

[0041] ⑥ProAtU6-26::sgRNA and CLCrV virus accumulation detection:

[0042] Since upland cotton is an allotetraploid plant with a large genome, the same gene may have naturally mutated SNP sites in the A / D subgroup, resulting in single-base editing false positive results. Using the true leaves of plants with target gene mutations and the genomic DNA of wild-type plants as templates, specific primers for amplifying the U6-sgRNA fragment were designed, as shown in Table 1. PCR amplification was used to detect whether the target gene sgRNA driven by the AtU6-26 promoter was expressed in the inoculated cotton true leaves. In addition, the accumulation of CLCrV-B virus was detected in the true leaves of plants with target gene mutations to determine the spread of CLCrV virus in the cotton system.

[0043] ⑦ Off-target detection:

[0044] While the 20bp sgRNA binds to the target site of the target gene, it may also bind to other sites in the genome, resulting in off-target phenomena. In order to detect the specificity of gene editing by the ABE system mediated by CLCrV, the CRISPR-GE online tool developed by the research group of Academician Liu Yaoguang of South China Agricultural University was used to retrieve potential off-target sites based on the designed sgGhPEBP-ABE sequence. Potential off-target sites were selected to be located in the CDS region, and three sites with off-score scores from high to low were selected. Each predicted off-target sequence contained four mismatched bases. These potential off-target sequences were BLAST-aligned in the upland cotton database to retrieve the corresponding genomic sequences. Off-target rate detection primers were designed, as shown in Table 1, and Hi-TOM high-throughput sequencing was used to detect the specificity of gene editing.

[0045] Example 2

[0046] ① Detection of nCas9 expression in different nCas9-TadA7.10-OE plants

[0047] The high expression of nCas9 is a key factor affecting the CLCrV-mediated ABE system to achieve target gene editing and produce mutant phenotypes. In order to detect whether nCas9 is stably expressed and expressed in different strains of VIGE receptor nCas9-TadA7.10-OE plants and whether there are differences in expression levels between different plants of the same strain. qPCR amplified the 231bp nCas9 gene fragment. The results showed that the nCas9 gene was stably expressed in different nCas9-TadA7.10-OE strains, and there were certain differences in expression levels between different strains, but there were no obvious differences between different plants of the same strain, such as Figure 1 As shown, in this example, the nCas9-1 strain with a relatively high expression level of nCas9 was selected for subsequent research.

[0048] ② Construction of CLCrV-ABEs vector

[0049] The candidate CLCrV-AtU6-26::sgGhCLA1-ABE1, CLCrV-AtU6-26::sgGhCLA1-ABE2, CLCrV-AtU6-26::sgGhPDS-ABE and CLCrV-AtU6-26::sgGhPEBP-ABE recombinant plasmids were identified by restriction digestion.

[0050] The results showed that after double digestion with SpeⅠ and PacⅠ, the above recombinant plasmids produced two bands, a 516bp AtU6-26::sgRNA-ABEs fragment and a 11282bp CLCrV-A vector fragment. The digestion results were consistent with the expected digestion patterns, such as Figure 2As shown, it was shown that the final vectors CLCrV-AtU6-26::sgG hCLA1-ABE1, CLCrV-AtU6-26::sgGhCLA1-ABE2, CLCrV-AtU6-26::sgGhPDS-ABE and CLCrV-AtU6-26::sgGhPEBP-ABE for targeted editing of cotton GhCLA1, GhPDS and GhPEBP genes had been successfully constructed.

[0051] ③Mutation detection and gene editing efficiency analysis

[0052] Mutation detection was performed on each nCas9-TadA7.10-OE plant inoculated with CLCrV-AtU6-26::sgGhCLA1-ABE1 (11 strains), CLC rV-AtU6-26::sgGhCLA1-ABE2 (15 strains), CLCrV-AtU6-26::sgGhPDS-ABE (12 strains), and CLCrV-AtU6-26::sgGhPEBP-ABE (14 strains).

[0053] Amplify the target gene fragment with the correct size and unique band in all inoculated plants, such as Figure 3 As shown. Hi-TOM high-throughput sequencing was performed on all the above PCR products, and the deep sequencing results showed that no mutations were detected at the target site of the two GhCLA1 (GhCLA1-1 and GhCLA1-2) and GhP DS genes, and the genotype was consistent with the wild type. However, 7 of the 14 plants inoculated with CLCrV-AtU6-26::sgGhPEBP-ABE had mutations, and the plants numbered #3, #6, #10 and #14 simultaneously showed base editing of the GhPEBP gene in the cotton A / D subgroup. Among them, the A bases at the 5th and 9th positions on the target sites of #3 and #6 mutated to G bases, with editing efficiencies of 29.26% and 26.33%, respectively, while only the A base at the 5th position on the target site was detected to mutate to G base in #10 and #14, with editing efficiencies of 26.17% and 22.06%, respectively.

[0054] In addition, #5 and #11 only detected mutations of the 5th A base in the GhPEBP target site on the A subgroup, with editing efficiencies of 6.18% and 12.75%, respectively. #9 detected mutations of the 5th and 9th A bases on the D subgroup, with an editing efficiency of 13.81%, as shown in Table 2. According to statistical analysis, the editing efficiency of the GhPEBP gene by the CLCrV-mediated ABE base editing system was between 6.18 and 29.26%.

[0055] Table 2 GhPEBP mutation detection

[0056]

[0057]

[0058] In the above table, WT means wild type and SNP means base substitution.

[0059] ④ Target site mutation genotype detection

[0060] In view of the fact that the embodiment is the result of transient transformation, the GhPEBP mutant plant should be a chimera, and therefore, overlapping peaks should appear at the GhPEBP mutation site. Since there is a difference of 2 bases in GhPEBP near the target sites of cotton subgroup A and subgroup D, specific primers (as shown in Table 1) were used to amplify the genomic sequences of GhPEBP-A and GhPEBP-B covering the target sites, and the #3 plant with the highest base editing efficiency was selected for Sanger sequencing. The sequencing peak graph results showed that in the wild-type material, GhPEBP-A and GhPEBP-B both presented a single peak, while in the #3 plant, an obvious overlapping peak appeared at the 5th A base on the GhPEBP target sequence in the A / D subgroup, showing a signal of the G base, but no overlapping peak was detected at the 9th A base on the target sequence, as shown in Figure 1. Figure 4 As shown, Figure 4 WT indicates wild type; M indicates GhPEBP mutant sequence; S represents serine (Ser); G represents glutamic acid (Glu); black horizontal bars and arrows indicate mutation sites. Since the fifth A base mutated to a G base, the original site serine (Ser) was converted to glutamic acid (Glu), achieving amino acid substitution.

[0061] ⑤ProAtU6-26::sgRNA and CLCrV virus accumulation detection

[0062] In order to further clarify that the GhPEBP mutant genotype was the result of gene editing, the genomic DNA of the true leaves of plants #3, #6, #10 and #14, where GhPEBP base editing occurred simultaneously in the A / D subgroup, was used as a template to amplify the ProAtU6-26::sgGhPEBP fragment (351 bp) using primers ProAtU6-26F and sgGhPEBP-ABER. The results showed that the AtU6-26 promoter drove the sgRNA of GhPEBP to be stably expressed in the true leaves of cotton, such as Figure 5 As shown in A. In addition, the accumulation of CLCrV-B virus was detected in the true leaves of plants #3, #6, #10 and #14. The results showed that the spread and accumulation of CLCrV-B were detected in the true leaves of the plants inoculated with CLCrV-AtU6-26::sgGhPEBP-ABE. Figure 5 As shown in B. The above results indicate that the mutant genotype produced by GhPEBP is indeed the result of base editing.

[0063] ⑥ GhPEBP-sgRNA off-target analysis

[0064] The CLCrV-mediated ABE base editing system can achieve GhPEBP gene editing, but whether this target has potential off-target effects still needs further verification. According to the off-score values ​​predicted in the CRISPR-GE online database, three potential off-target sites were retrieved, such as Figure 6 As shown, off-target detection was performed on plant #3 where GhPEBP mutation was detected. The results of deep sequencing (5000 reads for each potential off-target site) showed that no mutations were detected in the three potential off-target sites, as shown in Table 3 below, indicating that the CLCrV-mediated ABE base editing system has a certain gene editing specificity.

[0065] Table 3 Detection and analysis of potential off-target sites

[0066]

[0067] Example 3

[0068] Precise base editing technology can not only replicate discovered superior allelic variants in plants, but also artificially create superior allelic variants that do not exist in nature, showing great application potential in plant gene function research and accelerating crop genetic improvement. The traditional cotton base editing system requires the use of Agrobacterium and tissue culture to transform the complete CRISPR base editor into cotton embryonic callus to achieve target gene editing. However, due to genotype limitations, there are relatively few cotton materials that can actually achieve genetic transformation, which is an obstacle for cotton, which is difficult to genetically transform.

[0069] Studies have reported that virus-induced gene editing technology is expected to solve this technical problem. Some plant virus vectors are able to deliver complete CRISPR editing elements and can achieve heritable gene editing. Therefore, it is of great significance to develop more plant virus-mediated gene editing tools in cotton. Compared with some plant viruses with larger carrying capacity, CLCrV does not have the characteristics of delivering complete CRISPR / Cas components. In order to establish a CLCrV-mediated ABE base editing system in cotton, this example uses nCas9-TadA7.10-OE cotton as the VIGE receptor, and detects the expression level of nCas9 between different strains and different plants of the same strain. The results show that the nCas9 gene is stably genetically expressed in these transgenic strains, such as Figure 1As shown in Table 2, this provides the necessary experimental materials for verifying the effectiveness and efficiency of the CLCrV-mediated ABE base editing system. Four CLCrV-ABEs viral vectors were inoculated into nCas9-TadA7.10-OE cotton and mutation detection was performed. The high-throughput sequencing results showed that in all inoculated plants, except for the conversion of the 5th and 9th A bases to G bases on the GhPEBP gene target sequence, no mutations were detected at the other two gene target sites, as shown in Table 2.

[0070] It is known that sgGhCLA1-ABE1, sgGhCLA1-ABE2 and sgGhPDS-ABE are three sgRNAs that can recognize and bind to the target sites of the GhCLA1 and GhPDS genes. The reason why the editing of these three sites was not detected in this example may be that the editing window of the base editing system is narrower than that of the Cas9 system and has a certain position preference. Unlike the Cas9 system, if a frameshift mutation occurs at the target gene at the target site, a staggered peak will appear at the corresponding position, while the base editing system will have overlapping peaks at the corresponding base positions. In order to verify this conjecture, plants with relatively high editing efficiency were selected for Sanger sequencing to clarify the mutant genotype of GhPEBP. The sequencing peak graph results showed that an obvious overlapping peak appeared at the 5th A base of the GhPEBP gene target site on the cotton A / D subgroup, showing the signal of the G base, such as Figure 4 As shown, the result is consistent with the expected result. Due to the low editing efficiency of the 9th A base (2.53% to 3.48%), this example did not detect the phenomenon of overlapping peaks at this site, which is consistent with the high-throughput sequencing results, and it also proves that the base editing system does have a certain position preference. Continue to detect the expression of ProAtU6-26::sgGhPEBP and the accumulation and diffusion of CLCrV in the true leaves of the mutant plants. The results show that the expression of sgGhPEBP can be detected in the true leaves of the inoculated plants, and the accumulation and diffusion of CLCrV-B can be detected, as shown in Figure 5 As shown. Previous studies have reported that the DNA fragments inserted into the viral vector may be lost as the virus spreads in order to improve the adaptability of the virus in the host cells. However, this phenomenon was not detected in this example, which indirectly proves the integrity of the CLCrV-AtU6-26::sgGhPEBP-ABE vector during the infection of plants. The above evidence shows that the targeted mutation of GhPEBP is indeed the result of base editing. Studies have reported that the ABE base editing system can also induce cytosine deamination at the target site, thereby producing some gene editing byproducts. Therefore, this example continues to explore the gene editing specificity of the CLCrV-mediated ABE base editing system. The results show that no off-target phenomenon was detected at the predicted potential off-target sites, such as Figure 6As shown in Table 3, this system has a certain gene editing specificity.

[0071] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. Establishment of a plant virus-mediated ABE base editing system in cotton, characterized in that: The ABE base editing system uses CLCrV as a delivery medium and nCas9-TadA7.10-OE cotton as a transformation receptor to construct an ABE base editing system mediated by the plant virus CLCrV in cotton.

2. The establishment of a plant virus-mediated ABE base editing system in cotton according to claim 1, characterized in that: The ABE base editing system mediated by the plant virus CLCrV in cotton can achieve targeted mutation of the target gene (GhPEBP).

3. The establishment of a plant virus-mediated ABE base editing system in cotton according to claim 1, characterized in that: The editing efficiency of the ABE base editing system mediated by the plant virus CLCrV in the cotton for the GhPEBP gene is 6.18% to 29.26%.

4. The establishment of a plant virus-mediated ABE base editing system in cotton according to claim 1, characterized in that: The ABE base editing system mediated by the plant virus CLCrV in cotton has certain gene editing specificity.

5. An application of the establishment of a plant virus-mediated ABE base editing system in cotton as described in any one of claims 1 to 4, characterized in that: The ABE base editing system mediated by the plant virus CLCrV in cotton can be used for screening efficient sgRNAs in cotton and for cotton molecular design breeding.

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