Method for improving guide editing efficiency of dicotyledon

Abstract

The invention discloses a method for improving the guided editing efficiency of dicotyledonous plants, and the precise editing efficiency of the dicotyledonous plants is improved through a flank-Nicks Prime Editor (FLICK-PE). According to FLICK-PE, double notches are formed in the two sides of a target site, compared with PE3, the editing efficiency in soybeans can be improved by 4.8 times, and compared with PE2, the precise editing efficiency can be improved by 35.8 times. By utilizing an FLICK-PE strategy, TAP-IVS mutation is introduced into the GmEPSPS1a gene, a soybean stable transformation plant with glyphosate resistance is obtained, and a soybean gene editing plant with strong herbicide tolerance and basically unaffected agronomic characters is cultivated. The FLICK-PE can also realize accurate editing in tobacco, so that the FLICK-PE strategy can be used as a general tool and can be used for accurate breeding of important agricultural crops.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for improving the efficiency of guided editing in dicotyledons. Background Art

[0002] Precision gene editing tools are essential for targeted crop improvement strategies to address the challenges of modern agriculture. Primed editing systems based on the CRISPR / Cas system have become a transformative genome engineering technology, enabling precise base substitutions, insertions, and deletions. This system combines the functions of the Cas9 nickase (nCas9) with a reverse transcriptase (RT) enzyme, enabling precise integration of edits directly into the target genomic locus via a guide editing RNA (pegRNA) containing a primer binding site (PBS) and a RT template (RTT). Since the inception of this technology, extensive work has been devoted to improving its editing efficiency in plants. These optimizations include, but are not limited to, enhancing the thermostability and activity of the RT, modifying the secondary structure of the pegRNA, integrating a selection excision system, introducing a replacement system, and opening chromatin through the expression of hFTO. High primer editing efficiency has been achieved in monocotyledonous plants such as rice, maize, and wheat, enabling applications such as multiple primer editing, large fragment manipulation, and kilobase-scale DNA integration via primeRoot.

[0003] Unlike the rapid development of monocots, the implementation of guide-editing systems in dicots has been extremely challenging, with extremely low editing efficiencies (less than 1%) and difficulty in generating heritable edits. Currently, in tomato and Arabidopsis, precise insertion of heat-responsive elements into the invertase gene promoter has been achieved through combinatorial optimization strategies, including expression of guide-editing system components driven by strong constitutive promoters, designed epegRNA constructs, geminivirus replicon-mediated delivery, and post-transfection heat treatment. However, current progress remains insufficient for routine application in many important dicot crops, such as soybean. Therefore, in addition to leveraging classic strategies from mammalian systems and monocots, there is still a need to develop new optimization methods to improve guide-editing efficiency in dicots.

[0004] Manipulating the DNA mismatch repair (MMR) pathway can also improve targeting efficiency. In rice, overexpression of the dominant-negative variant MLH1dn or conditional inhibition of the key MMR component MLH1 significantly improved precision editing efficiency. The PE3 strategy introduces additional nicks on the non-edited strand, potentially inducing MMR to target the edited strand for repair, and exhibits up to fourfold greater efficacy than PE2 in human cells. In rice, some studies have shown limited efficiency improvements over PE2, but in some cases, it has also demonstrated higher activity. In dicots, some studies have introduced PE3, achieving editing efficiencies ranging from no heritable editing to over 10%. Therefore, the efficiency of nicking strategies varies across plant systems, potentially due to positional effects of different nicking sgRNAs, expression efficiencies of nicking sgRNAs, or differences in MMR preferences for different editing sites across different systems. The importance of nicking for targeted editing in plants, particularly in dicots, remains uncertain.

[0005] Existing technologies have low PE efficiency in dicots. Traditional optimization methods (such as engineering pegRNA, optimizing the expression of components in the guide-editing system, viral delivery, and heat treatment) can achieve precise editing in tomato and Arabidopsis. However, compared to monocots, these strategies are not routinely applicable in dicots. This suggests that endogenous factors beyond PE component optimization may limit the efficiency of guide-editing in dicots. To date, there is no efficient guide-editing tool for soybean. Therefore, further development and establishment of more efficient guide-editing technologies are needed to lay the technical foundation for soybean precision breeding, realize the application of guide-editing systems in soybean breeding, and create new germplasm. Summary of the Invention

[0006] The purpose of the present invention is to achieve efficient guided editing in dicotyledons. Based on this, a new PE system FLICK-PE is proposed.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: A guided editing system for improving the efficiency of guided editing in dicotyledons, wherein the system achieves the improvement of editing efficiency through one of the following strategies: (a) PE-NES nick : Introducing a nicking sgRNA into the PE2 system, the nicking sgRNA targets downstream of the non-editing strand pegRNA nick; (b) FLICK-PE: Two nicking sgRNAs were introduced into the PE2 system, targeting the upstream and downstream of the non-editing chain pegRNA nick, respectively.

[0008] Furthermore, the nicking sgRNA described in strategy (a) is located 20-80 bp downstream of the non-editing strand target.

[0009] Furthermore, the nicking sgRNA described in strategy (b) is located on both sides or on the same side of the non-editing strand within 100 bp upstream and downstream of the nicking site induced by the pegRNA.

[0010] Furthermore, the guide editing system includes: a) Expression of nCas9-reverse transcriptase fusion protein; b) at least one guide editing RNA comprising a primer binding site and a reverse transcription template for targeting a specific site in the target genome; c) At least one nicking sgRNA that targets the nicking site on the non-edited strand.

[0011] Furthermore, dicotyledonous plants include soybean, tobacco, Arabidopsis, and tomato.

[0012] Furthermore, the dicotyledonous plants are soybean and tobacco.

[0013] A method for improving the efficiency of guide editing in dicotyledons using the guide editing system described above comprises the following steps: a) Building PE-NES nick Or FLICK-PE boot editing system; b) introducing the guide editing system into a dicotyledonous plant cell; c) Screening and obtaining dicotyledonous cells or plants containing the target gene editing.

[0014] The gene editing is site-specific replacement, deletion or insertion.

[0015] The beneficial effects of the present invention are: The application of existing technologies in dicotyledons does not mention the use of nick sgRNA to improve PE efficiency. However, the novel strategy of the present invention using one or two nick sgRNAs can significantly improve PE efficiency in dicotyledons. In the present invention, PE3 (PE-NES nick) system can significantly improve the efficiency of guide editing in soybeans by introducing additional incisions, up to 11.8 times higher than the PE2 strategy. A flanking incision guide editor (FLICK-PE) system was further developed to introduce two incision sgRNAs on both sides of the target site on the non-editing strand, which improved the efficiency of PE editing in soybeans and tobacco by up to 4.8 times compared with the PE3 strategy and 35.8 times compared with the PE2 system. Using the FLICK-PE tool, heritable soybean lines with significant tolerance to glyphosate were developed in the laboratory and the field. FLICK-PE can also achieve precise editing in tobacco, highlighting its wide applicability. Therefore, the FLICK-PE strategy can be used as a general tool for precision breeding of important agricultural crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a classic optimization of the PE2 tool in soybean; (a) the vector structure of the main components of the SoyPE-V1 to SoyPE-V5 lead editors; (b) the design of pegRNA-1 to pegRNA-5 targeting 7 genes; (c) the percentage of precise editing efficiency of SoyPE-V1 to SoyPE-V5 at 7 sites in soybean hairy roots detected by Hi-TOM deep sequencing. The data in Figure c are from three biological replicates, involving a total of 20 to 30 hairy root samples; (d) the percentage of precise editing efficiency of SoyPE-V5 and SoyPE-V6 in the soybean hairy root system; (e) the percentage of precise editing of SoyPE-V6 in soybean stable transformation.

[0017] Figure 2 PE-ES in soybean hairy roots nick and PE3 (PE-NES nick ) strategies; (a) is precise editing mediated by endogenous cellular DNA repair; (b) is PE-ES nick Strategy Overview; (c) PE3 (PE-NES nick ) Strategy overview; (d)-(j) Detection of PE-ES by Hi-TOM deep sequencing nick and PE3 (PE-NES nick ) Percentage of precise editing efficiency at seven sites in soybean hairy roots, data from three biological replicates involving 20 to 30 hairy root samples; (k) PE3 (PE-NES nick ) is the fold change of the precise editing efficiency of the most effective vector relative to PE2 in 7 target sites; (l) is PE3 (PE-NES nick ) Correlation analysis between the position of sgRNA and precise editing efficiency; (m) is PE-NESnick Statistical analysis of strategy editing efficiency (calculated as the proportion of hairy roots with efficient editing among all tested roots).

[0018] Figure 3 Information on sgRNA design for seven target sites in soybean.

[0019] Figure 4 This is the pegRNA information in the soybean FLICK-PE strategy.

[0020] Figure 5 This is the nick sgRNA (Target-site-1~3) information in the soybean FLICK-PE strategy.

[0021] Figure 6 Nick sgRNA (Target-site-4~7) information in soybean FLICK-PE strategy.

[0022] Figure 7 Nick sgRNA (Target-site-8~11) information in soybean FLICK-PE strategy.

[0023] Figure 8 Evaluation of the PE efficiency of the FLICK-PE strategy in soybean hairy roots; (a) Overview of the design of the double-incision-guided editing strategy; Percentage of precise editing efficiency at target site 3 (b) and target site 5 (c) induced by three types of double-incision strategies in soybean hairy roots, the digital labels quantify the distance (in base pairs) between the unidirectional incision RNA cleavage site and the incision induced by the guide editing RNA (pegRNA), the plus (+) and minus (-) signs indicate the downstream or upstream position relative to the guide editing RNA cleavage site, respectively, and the letters F and R represent the editing strand or non-editing strand targeted by the single guide RNA, respectively; (d) Fold changes in the percentages of precise editing, imprecise editing, and indels at the two target sites Target-3 and Target-5 for all three double-nicking strategies compared with ESnick; percentages of precise editing, imprecise editing, and indels at Target-1 (e), Target-8 (f), Target-9 (g), Target-10 (h), and Target-11 (i) induced by FLICK-PE, PE-NESnick, and PE2 strategies in soybean hairy roots, data from three biological replicates involving 20 to 30 hairy root samples; (j) compared with PE-NESnick. nick Fold changes in the percentage of precise editing, imprecise editing, and indels of the FLICK–PE strategy on Target-1 and Target-8 to Target-11 compared; (k) FLICK-PE and PE-NESnick Effective precision editing efficiency in soybean hairy roots.

[0024] Figure 9 Design information for targets 8-11 in soybean.

[0025] Figure 10 To cultivate glyphosate-resistant soybeans using FLICK-PE technology and conduct phenotypic evaluation; (a) GmEPSPS1 Gene structure and epsps TAP-IVS Schematic diagram of mutation sites; (b) Hi-TOM deep sequencing to detect the mutations induced by FLICK-PE strategy in T0 generation soybean stable transformants GmEPSPS1 (Target-1) Percentages of precise editing, non-precise editing, and insertion / deletion; (c) Peak graph alignment of heterozygous T0 generation lines; (d) Genetic analysis of precise editing in T1 generation lines; (e) Peak graph alignment of homozygous T1 generation lines; After harvest epsps TAP-IVS Plant height (f), number of pods per plant (g), number of grains per plant (h), yield per plant (i) and 100-grain weight (j) of Huachun 6 (n = 30); (k) Mature Huachun 6 and epsps TAP-IVS Plant phenotypes; (l) Huachun 6 and epsps TAP-IVS Visualization of plant phenotypic responses to four concentrations of glyphosate.

[0026] Figure 11 FLICK-PE technology achieves precise editing in Nicotiana benthamiana; (a) Schematic diagram of the FLICK-PE-Nb vector structure; (b) Design of four pegRNAs in tobacco; (cf) Statistical diagram of the editing efficiency of six groups of vectors in tobacco transient transfection; (g) PE2, PE-NES nick Comparison of precise editing efficiency with FLICK-PE; (h) NbALS-1 gene structure and precise editing design; (ij) PE2 and PE-NES in tobacco stable transformation nick and FLICK-PE precise editing efficiency statistics; (kl) Sanger sequence comparison of T0 generation tobacco stable transgenic plants.

[0027] Figure 12 Target information for tobacco FLICK-PE strategy.

[0028] Figure 13 This is the pegRNA information for the tobacco FLICK-PE strategy.

[0029] Figure 14 This is the nick sgRNA information in the tobacco FLICK-PE strategy. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the present invention more clear and easy to understand, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific implementation described here is only for explaining the present invention and is not intended to limit the present invention.

[0031] Comparative Example 1 Classic Optimization of PE2 Tool First, we tested the effects of different optimized combinations of classical guide editing strategies on soybean guide editing efficiency (i.e., SoyPE-V1 to SoyPE-V5), including: 1) driving the endogenous soybean pGmM4 promoter to achieve efficient expression of the nSpCas9(H840A) variant (SoyPE-V1 to SoyPE-V5); 2) using nSpCas9-MMLV PEmax with stronger single-strand cleavage ability and nuclear localization (SoyPE-V3, SoyPE-V4, and SoyPE-V5); 3) testing the pGmU6 composite promoter (SoyPE-V2 and SoyPE-V3), pGmM8L (SoyPE-V4), and pGmUBQ3 (SoyPE-V5) strong promoters to enhance pegRNA transcription; and 4) testing single transcripts driven by pM4 to achieve coordinated expression of nCas9-RT and pegRNA (SoyPE-V1). The vector structure is shown in Figure 5. Figure 1 a. Subsequently, seven loci in soybean (pegRNA-1 targeting EPSPS1a ( Glyma.01g139600 ), pegRNA-2 targeting LOX3 ( Glyma.15G026300 ), pegRNA-3 targeting 7Sα1 ( Glyma.20G148400 ), pegRNA-4 targeting RIN1a ( Glyma.12G224600 ) and pegRNA-5 targeting three homologous genes: 11S-1 ( Glyma.03G163533 ), 11S-2 (Glyma.19G164900), and 11S-3 (Glyma.03G163467), a guide editing vector was designed and its efficiency was tested in the soybean hairy root system. Target details are shown in Figure 1 b. Hi-Tom deep sequencing revealed that the precision editing efficiency of these five tools was very low. The proportion of precision editing reads in each transgenic event did not exceed 19.52% of the total sequencing reads, and no PE tool could detect editing in all seven genes simultaneously. The results are shown in Figure 1c. Among these tools, SoyPE-V5, with PEmax driven by pGmM4 and pegRNA driven by pGmUBQ3, showed relatively high editing efficiency, with the highest percentage of precise editing at six sites ranging from 6.23% to 19.52%. SoyPE-V3, with pegRNA driven by the pGmU6 composite promoter, performed slightly worse than SoyPE-V5. Therefore, optimizing nCas9-RT and pegRNA expression can help achieve detectable precise editing in soybean, but the efficiency is still quite low. These methods have not yet been able to achieve heritable precise editing in stable transgenic soybean lines.

[0032] Example 1: Introduction of nicking sgRNA can significantly improve editing efficiency in soybean DNA repair mechanisms may prefer to use unedited sequences rather than the edited strands after reverse transcription editing as templates, which makes guided editing more difficult in soybeans. It is hypothesized that during the guided editing process in soybeans, the edited strands cannot be effectively integrated, thus limiting the occurrence of editing ( Figure 2 a) If the 3' flap of the edited strand fails to integrate effectively, the genome will eventually revert to the wild type (top). In contrast, if integration is successful, precise guided editing is achieved (bottom). Next, we tested whether competition from the original sequence on the edited strand (called the 5' flap) limits the integration of the newly synthesized edited strand in soybean. Therefore, the present invention designed a strategy to introduce an additional nick downstream of the target site of the edited strand, which may enhance the cutting of the 5' flap (the strategy is named PE-Edited-Strand-Nick, or PE-ES nick )( Figure 2 b). In the PE3 strategy ( Figure 2 c) Introducing the nick sgRNA at the non-edited strand (PE-NES nick ) has been proven effective in mammalian cells, but the results vary in different plant studies, which may be related to the position effect of the incision. Therefore, we compared the effectiveness of PE3 and PE2 in soybean and designed multiple incision sgRNAs for the above 7 sites based on the SoyPE-V5 vector. For each target site, PE-ES nick and PE3 (PE-NES nick ) designed 1-3 nicking sgRNAs respectively, and the target sgRNA design can be found in Figure 3 The nicking sgRNA is located 60-85 base pairs (bp) downstream of the target site. Compared with the PE2 strategy, except for Target-7, PE-ES nick There is no significant effect on PE efficiency ( Figure 2dj). In contrast, all designed PE3 (PE-NES nick ) vectors significantly improved editing efficiency. The editing efficiency of each transgenic event was 4.7 to 11.8 times higher than that of the PE2 system without nicking ( Figure 2 k). For example, at Target-1, the PE3 vector with a nick (+65bp) achieved the highest precise editing rate of 54.9% ( Figure 2 d). At Target-2, the PE3 vector with a nick (+27bp) achieved the highest precise editing rate of 76.4% ( Figure 2 e). At Target-3, PE3 vectors with cuts (+48bp) and (+67bp) achieved precise editing rates of 79.0% and 44.1%, respectively ( Figure 2 f). At Target-4, the PE3 vectors with cuts (+56bp) and (+68bp) achieved 65.2% and 39.4% precision rates, respectively ( Figure 2 g). For pegRNA-5 targeting three homologous genes, all PE3 vectors targeting these genes achieved efficient editing, while the control vector with a non-targeting nicking sgRNA (where the nicking sgRNA sequence contained a single nucleotide polymorphism (SNP) that could not target the gene) did not produce efficient editing ( Figure 2 hj).

[0033] The present invention also found that most effective nicking sgRNAs were designed 40-70 bp downstream of the target site ( Figure 2 i). Statistical analysis of the editing efficiency of the PE3 strategy (calculated as the proportion of hairy roots with effective editing among all tested roots) showed that among the seven target genes, the precise editing efficiency ranged from 8.33% to 34.9%, while varying degrees of non-precise editing and indels were present ( Figure 2 m). The present invention adopts a rationally designed nicking sgRNA for the PE3 strategy to achieve efficient editing in soybean hairy roots and significantly improve the editing efficiency.

[0034] Example 2 Application of FLICK-PE strategy in soybean Given that nicking on the non-editing strand significantly improves the efficiency of PE3, it is hypothesized that the limited guide editing efficiency in soybeans may be related to the fact that MMR prefers to use the non-editing strand rather than the editing strand as a repair template. Therefore, enhancing the instability of the non-editing strand by double nicking may further improve the editing efficiency of soybeans. To test this hypothesis, the present invention designed three new double nicking strategies based on the SoyPE-V5 vector: (1) Type I is designed as a flanking incision, that is, two unidirectional incision sgRNAs are located upstream and downstream of the non-editing strand target site, respectively, and are named flanking incision guide editor (FLICK-PE) (2) Type II (asymmetric incision) is designed as an asymmetric incision, that is, both incision sgRNAs are located downstream of the non-editing strand target site; (3) Type III (cross-strand incision): double-strand incision, that is, one incision is located in the editing strand and the other is located in the non-editing strand.

[0035] For more information on pegRNA and nick sgRNA, see Figure 4-Figure 7 Target-3 and Target-5 were selected to compare the editing efficiency of the three strategies. At these two sites, FLICK-PE achieved more efficient and precise editing compared with PE3 ( Figure 8 b-8d). In contrast, type II and type III strategies only increased the efficiency of non-precise editing and indels, while the efficiency of precise editing remained unchanged or decreased ( Figure 8 b-8d).

[0036] Considering that FLICK–PE showed strong editing ability while ensuring specificity, we selected this strategy for further evaluation. Five new target sites were designed, including Target-1 (previously used PE-NES nick strategy tested) and 4 new target sites (Target-8-11, see Figure 9 ). With PE-NES nick In comparison, FLICK-PE showed higher precision editing efficiency at the four tested target sites and performed comparable well at Target-10 ( Figure 8 The average percentage of precisely edited Hi-Tom reads per transgenic event was 1.6 to 4.8 times higher than that of the PE3 strategy, with an average of 2.2 times, and 8.7 to 35.8 times higher than that of the PE2 strategy, with an average of 15.7 times ( Figure 8 j). At Target-11, the PE strategy did not detect any effective editing, while FLICK-PE achieved precise editing with a success rate of 3.33% ( Figure 8 i). Statistical analysis of the effective precision editing efficiency of all seven tested target sites showed that the precision editing efficiency of FLICK–PE was increased by approximately 1.7 times on average compared to the PE3 strategy ( Figure 8 k). In summary, the FLICK-PE strategy using double-flanked sgRNAs on the non-editing strand significantly improved the efficiency of precise guide editing in soybean while maintaining specificity.

[0037] Example 3 Cultivation of glyphosate-resistant soybeans using FLICK-PE technology To test the ability of FLICK-PE in soybean breeding, a stable transformation experiment of Huachun 6 soybean variety was conducted. GmEPSPS1 TAP-IVS of the gene (Glyma.01G139600) was tested for guided editing ( Figure 10 a). Among the 46 transgenic T0 generation lines, 4 plants were identified by Hi-TOM detection with more than 23.46% effective precise editing reads (4 out of 46 plants) ( Figure 10 b). In addition, the editing status of these plants can be detected by Sanger sequencing, confirming that the proportion of precise editing has been significantly improved ( Figure 10 c). In the T1 generation, heritable homozygous edited plants were obtained ( epsps TAP-IVS ), which confirmed that FLICK-PE can achieve heritable editing in soybean stable transformation ( Figure 10 d, 10e).

[0038] In rice, carrying homozygous EPSPS-TAP-IVS Although the mutant plants are glyphosate tolerant, they exhibit severe growth defects and sterility. GmEPSPS1-TAP-IVS The plants were fertile, and statistics on plant height, number of pods, number of seeds per plant, yield per plant, or 100-seed weight showed that their growth was only slightly affected ( Figure 10 fk).

[0039] In field trials, glyphosate herbicide was tested at four concentrations: 1 (1.23 kg a.i. / ha), 2 (2.46 kg a.i. / ha), 4 (4.92 kg a.i. / ha), and 8 (9.84 kg a.i. / ha) times the applied concentration. GmEPSPS1-TAP-IVS Glyphosate resistance in plants. Wild-type plants died at all concentrations 10 days after spraying (applied 14 days after germination). In contrast, GmEPSPS1-TAP-IVS Plants showed no adverse effects at 2 times the concentration. At higher doses, GmEPSPS1-TAP-IVS Plants exhibit leaf curling and wilting symptoms, but remain viable and fertile ( Figure 10 1). The present invention successfully utilizes FLICK-PE technology to cultivate new soybean germplasm that is resistant to high doses of glyphosate without affecting soybean fertility.

[0040] Example 4 FLICK-PE induces efficient and precise editing in tobacco Based on the experimental results in soybean, in order to verify whether FLICK-PE is applicable to other dicotyledonous plants, we then tested it in tobacco ( Nicotianatabacum ) was tested. By constructing a tobacco-optimized PE carrier system FLICK-PE-Nb ( Figure 11a), in which the 35S enhanced promoter drives the expression of the nCas9-MMLV PEmax fusion protein. The AtU6 composite promoter drives the expression of pegRNA and nick sgRNA. In addition, an eGFP fluorescent marker is integrated for efficient transgenic screening. A total of six vectors were designed, including combinations of pegRNAs and different nick sgRNAs targeting four sites, covering small fragment insertions, deletions, substitutions, and their combinations ( Figure 11 b) and tested its functionality by transiently transforming leaf epidermal cells.

[0041] The design information of tobacco targets, pegRNAs, and nick sgRNAs can be found in Figure 12-14 Overall, the PE3 strategy improved the precision editing efficiency in all tested targets. At target site 1, in the two sets of vector designs based on the sgRNA targeting site, the precision editing efficiency of the vectors using the FLICK-PE strategy was significantly higher than that of their corresponding PE3 vectors, reaching 16.51% and 11.97%, respectively, which were 1.21 times and 1.96 times higher than those of PE3 ( Figure 11 c). At target site 2, the precise editing efficiency of the FLICK-PE vector in both vector groups was significantly higher than that of the PE3 vector, at 17.38% and 17.77%, respectively, which were increased by 1.77 times and 1.13 times, respectively ( Figure 11 d). At target site 3, the precise editing efficiency of FLICK-PE was slightly higher than that of PE3 vector (37.24% vs. 34.33%) ( Figure 11 e). At target site 4, the precise editing efficiency of FLICK-PE (8.02%) was significantly higher than that of PE3 (4.54%) ( Figure 11 f). Among all the 6 vectors tested, FLICK-PE significantly outperformed PE3, with improvements ranging from 1.08-fold to 1.96-fold (average: 1.49-fold) ( Figure 11 g).

[0042] PE-NES nick The FLICK-PE strategy has improved the precise editing efficiency of tobacco leaf transient transfection. Compared with PE, the FLICK-PE strategy has achieved further improvement in precise editing efficiency ( Figure 11 ), while PE2 failed to produce detectable editing. These findings indicate that our FLICK-PE strategy maintains robust efficacy in tobacco.

[0043] In subsequent tests using a stable transformation system, the tobacco ALS1 gene was selected for precise editing (consistent with target site 1 in the transient transfection system), and the W504L amino acid substitution was successfully achieved ( Figure 11h). HI-TOM sequencing analysis showed that PE2 failed to achieve efficient and precise editing, while FLICK-PE achieved an editing efficiency of 19.4%, exceeding PE3 (12.5%) ( Figure 11 i). Sanger sequencing verification further confirmed that the effectively edited samples showed double peaks in the sequencing peak graph. ( Figure 11 j). These findings indicate that FLICK-PE has a strong efficacy in tobacco.

[0044] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description should not be considered as limiting the present invention. After reading the above description, various modifications and substitutions of the present invention will be apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A guide editing system for improving the efficiency of guide editing in dicotyledons, characterized by: The system improves editing efficiency through one of the following strategies: (a) PE-NES nick : Introducing a nicking sgRNA into the PE2 system, the nicking sgRNA targets downstream of the non-editing strand pegRNA nick; (b) FLICK-PE: Two nicking sgRNAs are introduced into the PE2 system, wherein the nicking sgRNAs target the upstream and downstream of the non-editing chain pegRNA respectively or both nicks are located downstream.

2. The guide editing system according to claim 1, wherein: The nicking sgRNA described in strategy (a) is located 20-80 bp downstream of the nicking of the target pegRNA on the non-editing strand.

3. The guide editing system according to claim 1, wherein: The nicking sgRNA described in strategy (b) is located on both sides or on the same side of the non-editing strand within 100 bp upstream and downstream of the nicking site induced by the pegRNA.

4. The guide editing system according to claim 1, wherein: The guide editing system includes: a) Expression of nCas9-reverse transcriptase fusion protein; b) at least one guide editing pegRNA, which contains a primer binding site and a reverse transcription template for targeting a specific site in the target genome; c) At least one nicking sgRNA that targets the nicking site on the non-edited strand.

5. The guide editing system according to claim 1, wherein: Dicots include soybean, tobacco, Arabidopsis, and tomato.

6. The guide editing system according to claim 5, characterized in that: The dicotyledonous plants are soybean and tobacco.

7. A method for improving the efficiency of guide editing in dicotyledons using the guide editing system according to any one of claims 1 to 6, characterized in that: The following steps are involved: a) Building PE-NES nick Or FLICK-PE boot editing system; b) introducing the guide editing system into a dicotyledonous plant cell; c) Screening and obtaining dicotyledonous cells or plants containing the target gene editing.

8. The method according to claim 7, wherein: The gene editing is site-specific replacement, deletion or insertion.

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