A pea single-base editing system and application thereof in creating herbicide-resistant pea germplasm

Abstract

The application discloses a pea single-base editing system and application thereof in creating herbicide-resistant pea germplasm, and belongs to the technical field of plant genetic engineering and molecular breeding. The application provides a construction method of adenine base editors, cytosine base editors and double single-base editors suitable for peas, and each functional element of the system is codon-optimized for peas. The application also establishes a high-throughput resistance site screening method based on a PsALS gene saturation mutation library, and in combination with Hi-TOM high-throughput sequencing, quickly identifies key amino acid sites that confer herbicide resistance. On this basis, the application uses a multi-target single-base editing vector or a double single-base editing vector to create new pea germplasm resistant to imidazolinone and other ALS inhibitor herbicides through stable genetic transformation mediated by agrobacterium. The application fills the technical gap in the field of pea base editing, and provides an important tool and germplasm resource for precise breeding of peas.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and molecular breeding technology, specifically relating to a method for constructing a highly efficient adenine base editor (ABE), cytosine base editor (CBE), and double single base editor (A&C Twin-BE) for peas, and a method for using the above systems in combination with a PsALS gene saturated mutant library to screen for key amino acid sites resistant to herbicides and create new herbicide-resistant pea germplasm. Background Technology

[0002] Peas (Pisum sativum L.) are one of the world's most important edible legume crops, rich in protein, dietary fiber, and various vitamins, playing a vital role in food security, livestock feed, and vegetable supply. However, weed infestation is a serious problem during pea cultivation, competing with peas for water, nutrients, and sunlight, severely impacting pea yield and quality. Currently, there is a lack of herbicide-resistant pea varieties, the use of limited herbicides, and the increasing prevalence of herbicide-resistant weeds have become significant factors restricting the development of the pea industry.

[0003] Base editing is a novel, precise gene editing technology developed based on the CRISPR / Cas9 system. It allows for the precise replacement of single bases directly on genomic DNA without causing double-strand breaks (DSBs). Base editors mainly fall into two categories: adenine base editors (ABE), which convert A·T to G·C; and cytosine base editors (CBE), which convert C·G to T·A.

[0004] Currently, base editing technology has been successfully applied in various crops, including rice, wheat, corn, soybeans, and tomatoes. However, the development of base editing systems in peas is still lacking, which severely limits the study of gene function and genetic improvement of important agronomic traits in peas.

[0005] Acetolactate synthase (ALS; also known as acetylhydroxy acid synthase, AHAS) is a key enzyme in the biosynthesis of branched-chain amino acids (leucine, isoleucine, and valine). ALS inhibitor herbicides (including imidazolinones, sulfonylureas, and triazines) inhibit ALS enzyme activity, leading to the inhibition of branched-chain amino acid synthesis and plant death. Mutations at specific amino acid sites in the ALS gene can reduce the sensitivity of the enzyme protein to herbicides, thus conferring herbicide resistance to plants. In other crops (such as rice, wheat, soybeans, and corn), several ALS gene mutation sites have been reported to confer herbicide resistance, but these sites are mostly patented by foreign companies or research institutions, limiting their application. Herbicide resistance sites in the ALS gene of peas have not yet been reported.

[0006] The existing technology has the following shortcomings: (1) There is no mature base editing system in peas, which limits the progress of pea functional genomics research and precision breeding; (2) Most of the known ALS herbicide resistance sites are protected by patents and cannot be freely used for herbicide-resistant pea breeding in my country; (3) There is a lack of high-throughput resistance site screening methods for peas based on endogenous gene saturated mutation libraries; (4) The efficiency of pea stable genetic transformation system is low, and traditional gene knockout or transgenic methods are difficult to achieve precise amino acid replacement. Summary of the Invention

[0007] Purpose of the invention

[0008] This invention aims to solve the technical problems of the lack of efficient base editing systems for peas and the scarcity of herbicide-resistant pea germplasm in the prior art, and to provide an efficient single-base editing system suitable for peas and its application in the creation of herbicide-resistant pea germplasm.

[0009] Technical solution

[0010] In a first aspect, the present invention provides a single-base editing system suitable for peas, the system comprising an adenine base editor (ABE) and / or a cytosine base editor (CBE).

[0011] The adenine base editor comprises a first fusion protein, which, from the N-terminus to the C-terminus, comprises: an SV40 nuclear localization signal (NLS), TadA-8e adenine deaminase, a first linker peptide, a protein, and a 3×SV40 nuclear localization signal.

[0012] The cytosine base editor comprises a second fusion protein, which, from the N-terminus to the C-terminus, comprises: an SV40 nuclear localization signal, an A3A cytosine deaminase, a second linker peptide, an nCas9 (D10A) protein, a uracil glycosylation inhibitor (UGI), and a 3×SV40 nuclear localization signal.

[0013] The amino acid sequence of the nCas9(D10A) protein is shown in SEQ ID NO.1, and the amino acid sequence of TadA-8e adenine deaminase is 166 amino acids (sequence shown in SEQ ID NO.2). The first linker peptide is a flexible linker peptide of 32 amino acids (sequence shown in SEQ ID NO.5). The amino acid sequence of A3A cytosine deaminase is 199 amino acids (sequence shown in SEQ ID NO.3). The second linker peptide is an XTEN linker peptide (sequence shown in SEQ ID NO.6). The uracil glycosylase inhibitor UGI is derived from Bacillus subtilis phage PBS1 (amino acid sequence shown in SEQ ID NO.4).

[0014] Preferably, the coding sequences of the nCas9(D10A) protein, TadA-8e adenine deaminase, A3A cytosine deaminase, and uracil glycosylation inhibitor UGI have all been optimized using pea codons. The optimization principle is to prioritize the use of codons with a synonymous codon frequency of ≥30% in peas, and to remove AT-rich regions, cryptic splice sites, and internal repetitive sequences.

[0015] Preferably, the adenine base editor is driven by an enhanced promoter en35s, which is composed of a TM2 enhancer (an Omega sequence from the TMV virus), a pd35s promoter fragment (a CaMV 35S promoter fragment containing dual enhancers), and a dMac3 translational enhancer.

[0016] Preferably, the system further includes an sgRNA expression cassette, which is transcribed by the pea endogenous U6 promoter PsU6.3. The sgRNA expression cassette contains a tRNA-sgRNA fusion expression unit, wherein the tRNA is Arabidopsis tRNA-Gly, which can enhance the processing and stability of sgRNA.

[0017] In a second aspect, the present invention provides a dual-single-base editor (A&C Twin-BE) that can simultaneously perform A·T to G·C conversion and C·G to T·A conversion in the same cell.

[0018] The dual monobase editor comprises a third fusion protein, which is selected from one of the following structures:

[0019] Structure I (N-terminal double fusion): From N-terminus to C-terminus, it contains: SV40 nuclear localization signal, TadA-8e adenine deaminase, Flexible linker peptide, A3A cytosine deaminase, XTEN linker peptide, nCas9 (D10A) protein, uracil glycosylation inhibitor UGI, and 3×SV40 nuclear localization signal;

[0020] Structure II (N-terminal + C-terminal separation and fusion): From N-terminus to C-terminus, it contains: SV40 nuclear localization signal, TadA-8e adenine deaminase, XTEN linker peptide, nCas9 (D10A) protein, A3A cytosine deaminase, Flexible linker peptide, uracil glycosylation inhibitor UGI, and 3×SV40 nuclear localization signal.

[0021] Preferably, the uracil glycosylation inhibitor UGI comprises one copy of UGI and three vials. Flexible linker peptide tandem free UGI copy.

[0022] In a third aspect, the present invention provides a recombinant expression vector comprising the coding sequence of the aforementioned single-base editing system or double single-base editor.

[0023] Preferably, the recombinant expression vector further includes the Bar gene as a plant selection marker (conferring glufosinate resistance to transformed plants) and the SpR gene (spectruminant / streptomycin resistance gene) as a microbial selection marker.

[0024] Preferably, the recombinant expression vector contains left and right boundary sequences of T-DNA, mediating the transfer and integration of T-DNA.

[0025] In a fourth aspect, the present invention provides a pea sgRNA library containing multiple sgRNAs targeting the pea PsALS gene family, wherein the PsALS gene family includes three members: PsALS1, PsALS2, and PsALS3.

[0026] The sgRNA library was obtained using the following method: The CDS sequences of the pea PsALS1, PsALS2, and PsALS3 genes were used as input. A C language program was used to screen all sgRNA target sites with NGG PAM. Screening criteria included: the ABE editing window (positions 4-8 distal to the PAM) containing editable A bases and / or the CBE editing window (positions 3-9 distal to the PAM) containing editable C bases; removal of sgRNA sequences containing more than 4 consecutive T bases (Pol III termination signal); removal of sgRNA sequences causing synonymous mutations; and deduplication of redundant sgRNAs.

[0027] Preferably, the sgRNA library contains eight sgRNAs targeting the PsALS1 gene, wherein Targets 1-4 are used for adenine base editing (ABE editing) and Targets 5-8 are used for cytosine base editing (CBE editing).

[0028] In a fifth aspect, the present invention provides a method for screening herbicide resistance sites in the PsALS gene of peas, comprising the following steps:

[0029] (1) Constructing an sgRNA library targeting the pea PsALS gene family;

[0030] (2) The sgRNA library is cloned into the above recombinant expression vector to obtain the sgRNA library plasmid;

[0031] (3) The sgRNA library plasmid was transformed into Agrobacterium rhizogenes K599 to infect pea explants and induce the production of transgenic hairy roots;

[0032] (4) The hairy roots were cultured on a screening medium containing an acetolactate synthase ALS inhibitor herbicide to screen for resistant hairy roots;

[0033] (5) Genomic DNA was extracted from resistant hairy roots and the sgRNA target sequence and PsALS gene editing site were identified by sequencing;

[0034] (6) Identify the key amino acid sites of the PsALS gene that confers herbicide resistance.

[0035] Preferably, the ALS inhibitor herbicide is selected from imidazolinone herbicides (such as imidazolinone acetonide), sulfonylurea herbicides (such as chlorsulfuron-methyl and bensulfuron-methyl), or triazole pyrimidine herbicides (such as bispyribac-sodium).

[0036] In a sixth aspect, the present invention provides a method for creating herbicide-resistant pea germplasm, comprising the following steps:

[0037] (1) The key amino acid sites of the PsALS gene that confers herbicide resistance were screened according to the above method;

[0038] (2) Design and synthesize the corresponding sgRNA based on the key amino acid sites;

[0039] (3) Assemble one or more of the sgRNAs into the above recombinant expression vector to obtain a targeted editing vector; when assembling multiple sgRNAs, each sgRNA is separated by a tRNA sequence to form a tRNA-sgRNA tandem expression cassette.

[0040] (4) When the key amino acid site requires both A→G and C→T editing types, multiple sgRNAs are assembled into the above double monobase editor to obtain a multi-target double monobase editing vector.

[0041] (5) The targeted editing vector or the double single-base editing vector was transformed into Agrobacterium tumefaciens EHA105, and pea explants were transformed by Agrobacterium-mediated transformation.

[0042] (6) Screen positive transformants on a medium containing a screening agent (preferably glufosinate, concentration 3-10 mg / L), induce adventitious shoot differentiation, elongation and rooting, and obtain T0 generation regenerated plants;

[0043] (7) Molecular identification and herbicide resistance identification were performed on the T0 generation regenerated plants, and herbicide-resistant positive plants were screened;

[0044] (8) Self-pollinate positive plants to obtain genetically stable herbicide-resistant pea germplasm.

[0045] Preferably, the pea variety is Zhongwan 6.

[0046] Preferably, the herbicide resistance assessment includes spraying imidazolium acetonitrile at a concentration of 25-200 g ai / ha.

[0047] In a seventh aspect, the present invention provides herbicide-resistant pea germplasm created by the above method.

[0048] Preferably, the PsALS gene of the pea germplasm has been mutated at one or more of the following amino acid sites: positions 122, 197, 205, 574, and 653 corresponding to the ALS amino acid sequence of Arabidopsis thaliana.

[0049] Beneficial effects

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] (1) This invention establishes for the first time an efficient adenine base editing system and a cytosine base editing system in peas, filling the technological gap in the field of pea base editing and providing an important tool for pea functional genomics research and precision breeding.

[0052] (2) The dual single base editor (A&C Twin-BE) constructed in this invention can simultaneously achieve two types of base editing, A→G and C→T, in the same cell, which greatly expands the application scope of base editing.

[0053] (3) The present invention adopts a strategy of pea codon optimization, which significantly improves the expression efficiency and editing activity of base editor in pea cells.

[0054] (4) The enhanced promoter en35s developed in this invention has high transcriptional activity in pea cells, which is superior to the traditional CaMV 35S promoter.

[0055] (5) This invention establishes for the first time a high-throughput resistance site screening method based on a PsALS gene saturated mutation library, which can quickly and efficiently identify key amino acid sites that confer resistance to herbicides.

[0056] (6) The herbicide-resistant loci obtained by screening in this invention are original and provide usable gene resources for herbicide-resistant pea breeding in my country.

[0057] (7) The herbicide-resistant pea germplasm created in this invention provides important germplasm resources for the sustainable development of the pea industry. Attached Figure Description

[0058] Figure 1 Schematic diagram of the vector structure of the CRISPR / nCas9 single-base editing system.

[0059] A. The framework carrier pHUC-PsU6.3 SpR sg2.0-en35s PsCas9-t35sT structure, wherein en35s is an enhanced promoter en35s (TM2-pd35s-dMac3) that integrates TM2, pd35s and dMac3 elements.

[0060] B. The structure of the adenine base editor PsABE8e intermediate carrier;

[0061] C. The structure of the intermediate carrier of the cytosine base editor PsiA3A.

[0062] Figure 2 Identification of the PsALS gene family in peas and design of sgRNA target sites.

[0063] A. Schematic diagram of the gene structure and phylogenetic relationship of the three PsALS genes (PsALS1 / PsALS2 / PsALS3) in peas;

[0064] B. Schematic diagram of the locations of the 8 sgRNA target sites (Target 1-8) on the CDS of the PsALS1 gene; red bases (Target 1-4) represent the sgRNA target sequences constructed into the PsABE8e editor; green bases (Target 5-8) represent the sgRNA target sequences constructed into the PsiA3A editor; purple and brown represent PAM sites (NGG sequences).

[0065] Figure 3 Schematic diagram of the target vector structure of the CRISPR / nCas9 single-base editing system.

[0066] A. Schematic diagram of the target vector PsABE8e-tRNA-sgRNA;

[0067] B. Schematic diagram of the target vector PsiA3A-tRNA-sgRNA.

[0068] Figure 4 Roadmap for PsALS gene saturation mutation library construction and resistance screening.

[0069] Figure 5 Roadmap for the creation of new herbicide-resistant pea germplasm. Detailed Implementation

[0070] The present invention will be described in detail below with reference to embodiments. However, it should be understood that the following embodiments are merely illustrative examples of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. Unless otherwise specified, the experimental methods involved in the present invention are all methods commonly used in the art; the experimental reagents, instruments, etc. involved are all commercially available products unless otherwise specified, and should be operated according to the instructions; the gene sequences involved are all publicly available sequences unless otherwise specified.

[0071] Example 1: Construction of a pea base editing system

[0072] 1.1 Construction of the expression carrier skeleton

[0073] Using the gene editing backbone vector pHUC-PsU6.3 SpR sg2.0-en35s PsCas9-t35sT ( Figure 1 A) is the basis. The vector is approximately 15.2 kb in length and maintains high copy replication in E. coli DH5α.

[0074] The carrier contains the following elements:

[0075] (1) sgRNA expression unit (PsU6.3::tRNA-sgRNA-PolIII T, sequence as shown in SEQ ID NO.8): The pea 3 endogenous U6 promoter (PsU6.3, 487 bp in length, located in the scaffold_3 region of the pea genome, sequence as shown in SEQ ID NO.9) drives the transcription of the tRNA-sgRNA fusion expression cassette. The sgRNA backbone sequence is: 5'-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-3' (the sgRNA backbone termination signal is the Pol III terminator: 6 consecutive T bases, sequence as shown in SEQ ID NO.10). The tRNA (sequence as shown in SEQ ID NO.11) is Arabidopsis tRNA-Gly, which can enhance the processing and stability of sgRNA.

[0076] (2) Base editor protein expression unit (en35s::editor protein-t35s): The enhanced promoter en35s is composed of three elements: the TM2 enhancer (Omega sequence from TMV virus), pd35s (CaMV 35S promoter fragment containing dual enhancers), and the dMac3 translational enhancer (TM2-pd35s-dMac3), with a total length of approximately 1.2 kb (sequence shown in SEQ ID NO.7). The terminator is t35s (CaMV 35S polyA signal, approximately 220 bp, sequence shown in SEQ ID NO.12).

[0077] (3) Pea codon-optimized Cas9 (Kozak-SV40 NLS-SpCas9-3×SV40 NLS, sequence shown in SEQ ID NO.13): The SpCas9 protein coding sequence was optimized using the pea codon preference database, the GC content was adjusted to 52.5%, and hidden splice sites and polyA signals were removed. The Kozak sequence (5'-GCCACC-3') was added before the start codon ATG, SV40 NLS (amino acid sequence: PKKKRKVEDPKKKRKV) was fused to the N-terminus, and three tandem copies of SV40 NLS were fused to the C-terminus. The nuclear localization signals were linked by a GGGGS flexible linker.

[0078] (4) Selection markers: Bar gene (driven by CaMV 35S promoter) and SpR gene (aadA).

[0079] The Bar gene (from Streptomyces hygroscopicus, encoding glufosinate acetyltransferase) is driven by the CaMV35S promoter, conferring phosphinothricin resistance to transformed plants; SpR (spectinomycin / streptomycin resistance gene, aadA) is used for antibiotic screening in Escherichia coli and Agrobacterium. Recommended concentrations: spectinomycin 50-100 mg / L, streptomycin 25-50 mg / L, glufosinate 5-15 mg / L.

[0080] (5) T-DNA boundary sequences: Left Border / LB: 244 bp, sequence as shown in SEQ ID NO.14; Right Border / RB: 241 bp, sequence as shown in SEQ ID NO.15, containing a 25 bp core boundary repeat sequence, mediating T-DNA transfer and integration.

[0081] 1.2 Construction of intermediate vector for single-base editor

[0082] (1) nCas9 (D10A) site-directed mutagenesis

[0083] Using the pHUC-PsU6.3 SpR sg2.0-en35s PsCas9-t35sT vector as a template, PCR amplification was performed using site-directed mutagenesis primers to mutate codon GAT (Asp, position 10) to GCT (Ala).

[0084] Forward primer: 5'-CCTGAAGGCGAAGCTCCATGTGCTAC-3' (SEQ ID NO.16);

[0085] Reverse primer: 5'-GTAGCACATGGAGCTTCGCCTTCAGG-3' (SEQ ID NO.17).

[0086] PCR reaction system: template (pHUC-PsCas9) 50 ng (1 μL), forward primer (10 μM) 1.5 μL,

[0087] 1.5 μL of reverse primer (10 μM), 25 μL of 2×Phanta Max Master Mix (Vazyme), Add to a final volume of 50 μL.

[0088] PCR reaction program: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 sec for 18 cycles, annealing at 60℃ for 15 sec for 18 cycles, extension at 72℃ for 7 min / kb for 18 cycles, final extension at 72℃ for 10 min, and incubation at 4℃.

[0089] PCR products were verified by 0.8% agarose gel electrophoresis (containing 0.5 μg / mL EB substitute such as GoldView) in 1×TAE buffer at 5 V / cm for 30 min. Then, 1 μL of DpnI restriction endonuclease (NEB, 10 U / μL) was added, and the mixture was incubated at 37°C for 1 h to digest the methylated template plasmid. The DpnI-treated product was purified by ethanol precipitation (1 / 10 volume of 3M NaOAc (pH 5.2) and 2.5 volumes of anhydrous ethanol were added, precipitated at -20°C for 30 min, centrifuged at 12,000 rpm at 4°C for 10 min, washed once with 70% ethanol, air-dried, and dissolved in 20 μL of [amount not specified]. 5 μL of *E. coli* DH5α competent cells were transformed (50 μL, incubated on ice for 30 min, heat-shocked at 42°C for 45 sec, incubated on ice for 2 min, added to 500 μL LB liquid medium, incubated at 37°C 200 rpm for 1 h, plated on LB agar plates containing spectinomycin (50 mg / L), and incubated at 37°C for 16 h). A single colony was picked and inoculated into 5 mL LB liquid medium containing spectinomycin (50 mg / L), and incubated at 37°C 200 rpm for 14 h. Plasmids were extracted using the Vazyme FastPure Plasmid Mini Kit, and the D10A mutation was verified by Sanger sequencing (primer: 5'-CCTGAAGGCGAAGCTCCATGTGCTAC-3'). The correctly sequenced plasmid was named pHUC-PsU6.3 SpR sg2.0-en35s nCas9(D10A)-t35sT (abbreviated as pHUC-nCas9), and its sequence is shown in SEQ ID NO.18.

[0090] (2) Construction of PsABE8e, an intermediate vector for adenine base editor

[0091] Pea codon-optimized TadA-8e-32aa Linker gene synthesis

[0092] Pea codons were used to optimize the amino acid sequence of the adenine deaminase TadA-8e protein. The optimization principles were: prioritizing codons with a synonymous codon frequency ≥30% in peas; removing AT-rich regions (to avoid premature termination and low expression), cryptic splicing sites (predicted using NetGene2), internal repetitive sequences, and restriction enzyme sites (BsaI, BsmBI, BamHI, XbaI, SacI, etc., used for subsequent cloning were all removed). The optimized gene had a GC content of approximately 47%. The amino acid sequence of the TadA-8e-32aa linker is as follows:

[0093] TadA-8e (166 aa): MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDS (SEQ ID NO. 2).

[0094] 32aa Linker: SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO. 5).

[0095] The optimized TadA-8e-32aa Linker nucleotide sequence was synthesized by a biotechnology company into gBlocks gene fragments (500 ng / segment, with 15 bp homologous arms added at both ends for subsequent recombination).

[0096] Homologous recombination method to fuse TadA-8e-32aa Linker to the N-terminus of nCas9 (D10A)

[0097] Using the pHUC-nCas9 vector as a template, linearized primers were designed to amplify the vector backbone. Then, the TadA-8e-32aa linker was fused to the N-terminus of nCas9 (D10A) via homologous recombination. Positive clones were verified by colony PCR and Sanger sequencing. The correctly sequenced plasmid was named the PsABE8e intermediate vector. The specific process is as follows:

[0098] Using the pHUC-nCas9 vector as a template, a linearized primer amplification vector backbone was designed so that the target insertion site of the amplification product (the N-terminus of nCas9) has 15-20 bp homologous arms at both ends that overlap with the end of the TadA-8e-32aa Linker fragment.

[0099] Vector linearization primers: forward (extending backward from the start codon ATG): 5'-GGATCTTCTGGAGCTGGTTGTTCCGACGGCGAGCTACCTGAAGGAGCTGGAGCTCTAGACGCGTTCGAACC-3' (containing a homologous region with the C-terminus of TadA-8e) (SEQ ID NO.19); reverse (extending backward from downstream of the start codon ATG): 5'-ACCACTGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTAATAGTAAGTTAGTCAC-3' (SEQ ID NO.20).

[0100] XhoI linearized PCR system: pHUC-nCas9 template 50 ng (1 μL), primer F (10 μM) 1.5 μL, primer R (10 μM) 1.5 μL, 2× Phanta Max Master Mix 25 μL Bring the volume to 50 μL.

[0101] PCR program: 95℃ for 3 min; 95℃ for 15 sec, 60℃ for 15 sec, 72℃ for 7 min, for a total of 18 cycles; 72℃ for 10 min. The product was digested with DpnI (37℃, 1 h) to remove the template, and then recovered by gel electrophoresis with an elution volume of 30 μL. The concentration was determined by Nanodrop.

[0102] Homologous recombination reaction system (using ClonExpress II One Step Cloning Kit, Vazyme): linearized vector (approx. 50 ng / μL) 2 μL (~100 ng), TadA-8e-32aa Linker fragment (approx. 20 ng / μL) 2 μL (~40 ng), 5× CE II Buffer 4 μL, Exnase II 2 μL, Bring the volume to 20 μL.

[0103] Reaction conditions: Incubate at 37℃ for 30 min, then immediately cool on ice for 5 min. Transform 10 μL of the product into DH5α competent cells (transformation method as above), plate on LB agar plates containing spectinomycin (50 mg / L), and incubate at 37℃ for 16 h. Pick 4-6 single colonies and incubate in 5 mL LB liquid medium containing spectinomycin (50 mg / L) at 37℃ and 200 rpm for 14 h, then extract plasmids.

[0104] Colony PCR validation was performed using the following primer pairs (expected amplified fragment length: 894 bp with TadA-8e insertion, 300 bp with empty vector): Validation primer F: 5'-ATGTCTGAGGTGGAGTTTAGCCAC-3' (located at the 5' end of TadA-8e) (SEQ ID NO.21); Validation primer R: 5'-GAGATCGTCAGAAGCTTGCGC-3' (located in the N-terminal region of nCas9) (SEQ ID NO.22).

[0105] Colony PCR system (25 μL): 12.5 μL of 2× Taq Master Mix, 0.5 μL each of primers F / R (10 μM), and colony template (take a toothpick and stir it in the PCR tube after collecting the colony template). Bring the volume to 25 μL. PCR program: 95°C for 3 min; 95°C for 15 sec, 58°C for 20 sec, 72°C for 50 sec, for a total of 30 cycles; 72°C for 5 min. Positive clones were verified by 1.5% agarose gel electrophoresis (894 bp band appeared).

[0106] After extracting plasmids from positive clones, Sanger sequencing was performed using primer pair (F: 5'-ATGTCTGAGGTGGAGTTTAGCCAC-3' (SEQ ID NO. 23); R: 5'-CATGTGAGCTTGTCGGTCATGG-3' (SEQ ID NO. 24)) to verify the sequence correctness of the fusion site between the TadA-8e-32aa Linker and nCas9(D10A). The correctly sequenced plasmid was named the PsABE8e intermediate vector (pHUC-PsU6.3 SpR sg2.0-en35s TadA-8e-nCas9(D10A)-t35sT).

[0107] The intermediate carrier structure of PsABE8e is (from N-end to C-end): SV40 NLS - TadA-8e (166 aa) - 32 aa Linker (SGGSSGGSSGSETPGTSESATPESSGGSSGGSS) - nCas9(D10A) (1368 aa) - 3×SV40 NLS ( Figure 1 B), the sequence is shown in SEQ ID NO.25.

[0108] (3) Construction of PsiA3A, an intermediate vector for cytosine base editor

[0109] ① Synthesis of A3A-XTEN Linker and UGI+3×free UGI gene optimized by pea codons

[0110] Pea codons were optimized for the amino acid sequences of human APOBEC3A (A3A, 199 aa), XTEN Linker (32 aa), UGI (83 aa), and 3×free UGI (the optimization principles are the same as above).

[0111] The amino acid sequence of human APOBEC3A (A3A, cytosine deaminase domain, 199 aa) is: MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRYTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQNQGN (SEQ ID NO.3).

[0112] XTEN Linker amino acid sequence (32 aa): SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO.6).

[0113] UGI amino acid sequence (83 aa, derived from Bacillus subtilis phage PBS1): MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO.4).

[0114] 3×free UGI: Three UGI copies passed Flexible linker tandem (nucleotide sequence as shown in SEQ ID NO. 26).

[0115] Two independent gBlocks fragments were synthesized: Fragment A: A3A-XTEN Linker (798 bp, with 15 bp homologous arms added at both ends); Fragment B: UGI+3×free UGI (1,008 bp, with 15 bp homologous arms added at both ends).

[0116] ② Homologous recombination method: fuse A3A-XTEN Linker to the N-terminus of nCas9 (D10A), and UGI+3×free UGI to the C-terminus.

[0117] A two-round homologous recombination strategy was used: Round 1: fused UGI+3×free UGI to the C-terminus of nCas9. Using the pHUC-nCas9 vector as a template, primers were designed to linearize the vector (inserting UGI+3×free UGI before the nCas9 stop codon): the linearized PCR system was the same as above. The C-terminus of the amplified product had an overlapping arm homologous to the UGI+3×free UGI fragment. Homologous recombination reaction (20 μL, ClonExpress II system): 2 μL (~100 ng) of linearized vector (nCas9 C-terminus deleted) + 2 μL (~40 ng) of UGI+3×free UGI fragment. The reaction product was transformed into DH5α, and colony PCR was performed for verification (primer F: 5'-ATGAAGAACCTGCTGTTCGAGC-3' (SEQ ID NO.27), located at the 5' end of UGI, expected amplification of 1,008 bp), and Sanger sequencing was performed for verification. The intermediate vector pHUC-nCas9-UGI was obtained.

[0118] Round 2: Fusion of the A3A-XTEN Linker to the N-terminus of nCas9(D10A): Using pHUC-nCas9-UGI as a template, the A3A-XTEN Linker fragment was fused to the N-terminus of nCas9 after linearization, following the same method as above. Colony PCR verification (primers F: 5'-ATGGAGGCTTCTCCCGCTTCT-3' (A3A 5' end) (SEQ ID NO.28), R: 5'-CATGTGAGCTTGTCGGTCATGG-3' (nCas9 interior) (SEQ ID NO.29), expected fragment approximately 1,200 bp). The plasmid with the correct fusion sequence was sequenced and named the PsiA3A intermediate vector (pHUC-PsU6.3 SpR sg2.0-en35s A3A-nCas9(D10A)-UGI+3×free UGI-t35sT). The intermediate carrier structure of PsiA3A is as follows (from N-end to C-end): SV40 NLS - A3A (199 aa) - XTEN Linker (32 aa) - nCas9(D10A) (1368 aa) - UGI (83 aa) + 3×freeUGI (3×83 aa, via... Linker in series) - 3×SV40 NLS ( Figure 1 C), the sequence is shown in SEQ ID NO.30.

[0119] 1.3 Construction of a single-base editor targeting vector

[0120] (1) sgRNA target site design

[0121] Eight 20-nt target sites with a 5'-NGG-3' PAM sequence were selected within the CDS region of the pea PsALS1 gene (reference genome sequence number: Psat01G0547400). Targets 1-4 were used for the adenine base editor PsABE8e, and Targets 5-8 were used for the cytosine base editor PsiA3A.

[0122] The locations of the eight sgRNA target sites (Target 1~8) on the CDS of the PsALS1 gene are as follows: Figure 2 As shown in B. Red bases (Target 1-4) represent the sgRNA target sequences constructed into the PsABE8e editor; green bases (Target 5-8) represent the sgRNA target sequences constructed into the PsiA3A editor; purple and brown represent PAM sites (NGG sequences).

[0123] The sequences of the eight sgRNA target sites are as follows:

[0124] Target 1 (ABE-1) A→G CGAAAAACGTTGTTTGGGGT (SEQ ID NO.31) NGG Target 2 (ABE-2) A→G GACGGAAATGGGGTTTGGAG (SEQ ID NO.32) NGG Target 3 (ABE-3) A→G GTCGAAGCTCTCGAACGTCA (SEQ ID NO.33) NGG Target 4 (ABE-4) A→G ACTGATGTCTTTGCTTACCC (SEQ ID NO.34) NGG Target 5 (CBE-1) C→T CCACCAAGCACTCACGCGCT (SEQ ID NO.35) NGG Target 6 (CBE-2) C→T TTCCACCCTGTTCGTGACGG (SEQ ID NO.36) NGG Target 7 (CBE-3) C→T GCTCGATCCTCAGGCCTTCC (SEQ ID NO.37) NGG Target 8 (CBE-4) C→T GGTGCTACAAACCTCGTCAG (SEQ ID NO.38) NGG

[0125] (2) PCR amplification of tRNA-sgRNA expression cassette

[0126] The tRNA-sgRNA expression cassette was amplified using the plasmid pGTR-tRNA containing the tRNA (Gly) sequence as a template. The tRNA-Gly sequence (72 bp) was derived from Arabidopsis thaliana and could enhance the processing and stability of sgRNA.

[0127] PCR primer design: Add a BsaI restriction site and a homologous sequence of the tRNA forward primer to the 5' end of each sgRNA target site sequence, and add a BsaI restriction site and a reverse complementary sequence of the sgRNA backbone to the 3' end.

[0128] sgRNA primer design general rules: tRNA-F universal primer (SEQ ID NO.39): 5'-TGTGGTCTCAATTG(N)20GTTTTAGAGCTAGAAATAGC-3' (BsaI site GGTCTC is underlined, N20 is the target sequence, tRNA-tRNA-Gly and sgRNA backbone are fused by overlap extension PCR)

[0129] PCR reaction system: pGTR-tRNA template 10 ng (0.5 μL), tRNA-F (containing target sequence 10 μM) 1.5 μL, sgRNA-R universal primer (10 μM) 1.5 μL, 2× Phanta Max Master Mix 25 μL. Add to a final volume of 50 μL.

[0130] PCR program: 95℃ for 3 min; 95℃ for 15 sec, 58℃ for 30 sec, 72℃ for 30 sec, for a total of 30 cycles; 72℃ for 5 min. The product was verified by 1.5% agarose gel electrophoresis (expected ~330 bp), purified by gel recovery, and the elution volume was 30 μL.

[0131] (3) Golden Gate connection to construct a target vector

[0132] The tRNA-sgRNA PCR product was ligated into the PsABE8e or PsiA3A intermediate vector using the Golden Gate method. The Golden Gate reaction mixture consisted of: 2 μL (~200 ng) of PsABE8e or PsiA3A intermediate vector (approximately 100 ng / μL), 3 μL (~90 ng) of tRNA-sgRNA PCR product (approximately 30 ng / μL), 1.5 μL of 10× T4 DNA Ligase Buffer, 0.5 μL of BsaI-HFv2 (20 U / μL), 0.5 μL of T4 DNA Ligase (400 U / μL), and 0.15 μL of 10 mg / mL BSA. Add to 15 μL. Golden Gate cycling program (performed on a standard PCR instrument): 30 cycles of digestion-ligation at 37°C for 5 min, digestion at 50°C for 10 min, heat inactivation at 80°C for 10 min, and incubation at 4°C.

[0133] 5 μL of Golden Gate product was transformed into DH5α competent cells and plated on LB agar plates containing spectinomycin (50 mg / L), and incubated at 37°C for 16 h. Single colonies were picked for colony PCR verification (using sgRNA-specific primers and universal vector primers). Plasmids were extracted from positive clones and Sanger sequencing was performed to confirm the correctness of the sgRNA sequence. Sequencing primer (SEQ ID NO. 40): 5'-CGTTGTAAAACGACGGCCAGT-3' (located upstream of the sgRNA expression cassette).

[0134] The correctly constructed targeting vectors were named: PsABE8e-tRNA-sgRNA[X] (X represents ABE-1 to ABE-4, a total of 4 adenine base editing targeting vectors) and PsiA3A-tRNA-sgRNA[Y] (Y represents CBE-1 to CBE-4, a total of 4 cytosine base editing targeting vectors). Figure 3 This is a schematic diagram of the target vector structure for the CRISPR / nCas9 single-base editing system. Figure 3A. Schematic diagram of the target vector PsABE8e-tRNA-sgRNA, showing the assembly method of the tRNA-sgRNA expression cassette being attached to the intermediate vector. Figure 3 B. Schematic diagram of the target vector PsiA3A-tRNA-sgRNA structure, showing the assembly method of the tRNA-sgRNA expression cassette being attached to the intermediate vector.

[0135] Example 2: Identification of the editing capabilities of a single-base editing system

[0136] 2.1 Transformation of Agrobacterium rhizogenes K599 competent cells

[0137] Thaw 100 μL of K599 competent cells on ice, add 1 μL of plasmid DNA (approximately 200 ng), gently mix, and incubate on ice for 5 min. Transfer to a pre-chilled 2 mm electroporation cuvette (Bio-Rad Gene Pulser); electroporation parameters: 25 μF, 2.5 kV, 200 Ω (expected time constant 4-6 ms). Immediately add 1 mL of YEB liquid medium, gently mix, and transfer to a 1.5 mL centrifuge tube. Recover and culture at 28℃ and 200 rpm for 3 h. Spread 100 μL onto YEB solid plates (containing 50 mg / L spectinomycin + 25 mg / L streptomycin) and incubate at 28℃ for 48 h.

[0138] 2.2 Transient genetic transformation and hairy root induction in peas

[0139] The tested pea variety was Zhongwan 6, and healthy seeds with plumpness and uniform size were selected. Seed treatment and germination: Seeds were surface-sterilized in 75% ethanol for 30 seconds and rinsed once with sterile water; transferred to 3% (v / v) NaClO solution (containing 0.05% Tween-20), shaken for 10 min, and rinsed 5 times with sterile water; the sterilized seeds were then soaked in sterile water in the dark at room temperature (25°C) for 4-6 h; the seeds were then transferred to sterile filter paper and placed in a petri dish containing sterile water (seeds partially submerged), and incubated in the dark at 25°C for 2-3 days until germination (radicle length approximately 1-2 cm).

[0140] A single activated Agrobacterium K599 positive colony was inoculated into YEB liquid medium (containing 50 mg / L spectinomycin + 20 μM acetylsuccinone AS) and incubated at 28°C and 220 rpm until... =0.8-1.0. Collect bacterial cells by centrifugation at 5,000 rpm for 10 min at 4°C; ③ Resuspend in an equal volume of infection solution (liquid MS medium, pH 5.6, containing 100 μM AS, 0.02% Silwet L-77) and adjust. =0.6.

[0141] Take germinating pea seeds, remove the seed coat, and make shallow longitudinal cuts (3-4 cuts, about 1-2 mm deep, without cutting through) at the cotyledon nodes with a scalpel or make 2-3 cuts at the hypocotyl. Immerse the treated explants in K599 bacterial suspension and incubate at room temperature with shaking for 30 min (80 rpm). Remove the explants and blot off excess bacterial suspension with sterile filter paper. Transfer to co-culture medium (MS + 30 g / L sucrose + 8 g / L agar, pH 5.6, containing 100 μM AS) and incubate in the dark at 25°C for 3 days.

[0142] After co-culture, the explants were transferred to hairy root induction medium and cultured at 25°C under light (16 h light / 8 h dark, light intensity 50 μmol / m² / s). The medium was replaced with fresh medium every 10 days. After 3-4 weeks of culture, the hairy root development was observed and the hairy root induction rate was calculated as (number of explants producing hairy roots / total number of inoculated explants × 100%).

[0143] 2.3 Genomic DNA Extraction and Editing Efficiency Detection

[0144] Collect 50-100 mg of fresh hairy root tissue, grind it with liquid nitrogen, and extract genomic DNA. Design specific PCR primers (product length 400-600 bp) for each sgRNA target site, flanking sequences 200-300 bp on both sides of the target site.

[0145] PCR system: Template DNA (50 ng / μL) 2 μL (100 ng), forward primer F (containing Hi-TOM adapter, 10 μM) 1.5 μL, reverse primer R (containing Hi-TOM adapter, 10 μM) 1.5 μL, 2× KOD FX Neo Mix (TOYOBO) 25 μL, Bring the volume to 50 μL.

[0146] PCR program: 94℃ for 2 min; 94℃ for 30 sec, 58℃ for 30 sec, 68℃ for 40 sec, for a total of 35 cycles; 68℃ for 5 min. Products were verified by 2% agarose gel electrophoresis, and purified by gel extraction. PCR products were analyzed by Hi-TOM high-throughput sequencing.

[0147] Hi-TOM high-throughput sequencing analysis:

[0148] PCR products for each target site were mixed in equimolar amounts and Hi-TOM libraries were constructed using the TruePrep DNA Library Prep Kit (Vazyme). The libraries were sequenced using PE150 sequencing on an Illumina NovaSeq 6000 platform, with a target sequencing depth ≥10,000 reads / target site per sample. Data analysis was performed using the Hi-TOM online analysis platform (http: / / www.hi-tom.net / hi-tom / ) with the following parameters: window width 20 bp, minimum sequencing quality Q30, and matching accuracy ≥90%.

[0149] Editing efficiency parameter calculation:

[0150] Editing efficiency (%) = (Number of reads including those with target base editing / Total number of reads) × 100%

[0151] Edit purity (%) = (Number of reads with expected base editing / Total number of reads with editing events) × 100%

[0152] Edit window width: Taking the PAM position as a reference (the first nucleotide in PAM is +1), the editing frequency of each base position from the far end of PAM -1 to -17 (i.e. the 1st to 17th positions before N in the NGG sequence) is counted. Positions with an editing frequency >5% are defined as valid editing windows, and their start and end positions are recorded.

[0153] 2.4 Optimization of the base editor

[0154] Based on the detected editing efficiency of each target site, the base editor is iteratively optimized in multiple rounds:

[0155] First round of optimization (starter and NLS optimization): Compare the editing efficiency of en35s, CaMV 35S and pUbi10 under different starter drivers; compare different NLS configurations (N-end 1×SV40 NLS vs. 2×SV40 NLS vs. 1×SV40 NLS+1×c-Myc NLS, C-end 3×SV40 NLS vs. 2×SV40 NLS+1×bpNLS (double-fraction NLS)).

[0156] Second round of optimization (deaminase and linker optimization): ABE system to compare different variants of TadA-7.10, TadA-8e, and TadA-9; CBE system to compare different variants of A3A, A3G-CTD, eA3A, and evoCDA1; and comparison of different linkers of 32aa, 16aa, XTEN, and 3×GGGGS.

[0157] The third round of optimization (UGI optimization): compare the editing efficiency and purity of the edited product (C>T / C>G / C>A ratio) of 1×UGI, 2×UGI, 1×UGI+1×Gam (Mu Gam protein) and no UGI control.

[0158] After each round of optimization, the editing efficiency was tested. Three biological replicates were set up for each optimized variant, and ≥20 independent hair roots were detected in each replicate. The mean ± standard error was taken, and the statistical differences were compared using GraphPad Prism t-test (P<0.05, P<0.01, P<0.001).

[0159] Through the above rounds of optimization, a base editing system with the highest editing efficiency and best editing purity in pea cells was finally obtained.

[0160] Through multiple rounds of optimization, a base editing system with the highest editing efficiency and best editing purity in pea cells was obtained.

[0161] Example 3: Construction of a pea PsALS gene base editing library and screening of resistance sites

[0162] The technical route for constructing a PsALS gene saturation mutation library and screening for resistance is as follows: Figure 4 As shown.

[0163] 3.1 Bioinformatics Algorithm Design of sgRNA Libraries

[0164] A BLASTP search was performed on the pea reference genome using the alfalfa ALS protein sequence (MtALS, GenBank accession number: XP_003590727.1), soybean ALS protein sequence (GmALS, GenBank accession number: NP_001237638.2), rice ALS protein sequence (OsALS, GenBank accession number: XP_015628007.1), and maize ALS protein sequence (ZmALS, GenBank accession number: NP_001152852.1) as query sequences. The results revealed three ALS gene family members in the pea genome, located on two different chromosomes (Chr1 and Chr5), as detailed below:

[0165] PsALS1 Psat01G0547400 Chr1 1965 SEQ ID NO.41 PsALS2 Psat05G0170100 Chr5 1986 SEQ ID NO.42 PsALS3 Psat05G0170400 Chr5 1959 SEQ ID NO.43

[0166] All three genes contain only one exon (no introns), and their protein-coding sequences are highly conserved, with amino acid sequence identity ≥90% and CDS sequence identity ≥85%. Analysis of their motif distribution using the MEME online tool (https: / / meme-suite.org / meme / ) showed that the motif distributions of the three PsALS proteins were highly similar, further demonstrating the high conserved nature of their coding sequence domains. Figure 2 A).

[0167] PCR validation of the PsALS gene: Specific primers targeting the full-length CDS of the PsALS1, PsALS2, and PsALS3 genes were designed. Using genomic DNA from the 'Zhongwan 6' pea variety as a template, PCR amplification was performed using high-fidelity KOD FX Neo DNA polymerase. The PCR products were detected by 1% agarose gel electrophoresis, excised, and ligated into the pMD19-T vector (TaKaRa). Sanger sequencing was then performed, and the sequencing results were compared with the pea reference genome sequence. The results were consistent, confirming the accuracy of the reference genome sequence.

[0168] Write a C program for sgRNA library design. The algorithm logic is as follows: traverse each NGGPAM position of each CDS sequence, extract the 20 nt upstream sequence of the PAM as the sgRNA, calculate the A bases in the ABE editing window (positions 4-8 distal to the PAM) and the C bases in the CBE editing window (positions 3-9 distal to the PAM), infer the codon / amino acid changes after editing, and remove duplicate sgRNAs. The program outputs information such as the ID, target sequence, PAM, position, editing type, and expected amino acid substitutions for each sgRNA.

[0169] 3.2 Construction of sgRNA library plasmid

[0170] (1) Oligonucleotide microarray synthesis

[0171] The designed sgRNA target sequences (including the forward extension of tRNA and the reverse extension of the sgRNA backbone) were submitted to a biotechnology company for high-throughput Oligo microarray synthesis. Each Oligo microarray was approximately 120 nt in length (including a 20 nt target sequence and two constant flanking sequences), with a synthesis yield of >100 pmol / microarray and a synthesis scale of 12K or higher.

[0172] Oligo universal structure: 5'-TGTGGTCTCAATTG(N20)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTT-3' (SEQ ID NO.44), where the bolded part represents the N20 position of the target sequence and GGTCTC is the BsaI recognition site.

[0173] (2) Golden Gate Library Cloning

[0174] Linearize the PsABE8e or PsiA3A intermediate vector by digestion with BsaI-HFv2 (containing the ccdB suicide gene insertion site for screening).

[0175] Enzyme digestion system (50 μL): intermediate vector (1.5 μg) × μL, 10× CutSmart Buffer 5 μL, BsaI-HFv2 (20 U / μL), 1 μL Add to a final volume of 50 μL. Digest at 37°C for 2 h, and recover the linearized vector (removing a ~750 bp ccdB fragment) by 1% agarose gel electrophoresis, with an elution volume of 20 μL.

[0176] Oligo synthesized from microarrays was used as a template for PCR amplification and double-stranding. The double-stranded Oligo library was then cloned into a linearized vector using a Golden Gate library assembly reaction. Using the ccdB reverse selection system, only vectors with successfully inserted sgRNA were able to form colonies in E. coli.

[0177] Oligo library amplification and double stranding: Using microarray-synthesized Oligo as a template, PCR amplification was performed for 10 cycles using the following primers: Forward library primer: 5'-TGTGGTCTCAATTG-3' (containing the BsaI restriction site GGTCTC) (SEQ ID NO. 45), Reverse library primer: 5'-AAAAGCACCGACTCGGTG-3' (SEQ ID NO. 46). PCR system (50 μL, using KOD FX Neo): 1 μL of microarray Oligo template (~0.1 ng), 1.5 μL of each primer (10 μM), 25 μL of 2× KOD FX Neo Mix. Bring the volume to 50 μL. PCR program: 94℃ for 2 min; 94℃ for 30 sec, 55℃ for 20 sec, 68℃ for 20 sec, for a total of 10 cycles; 68℃ for 5 min. Purify the product using Agencourt AMPure XP magnetic beads (1.0× volume) to remove primer dimers, eluting in 20 μL.

[0178] Golden Gate library assembly reaction (50 μL large system): BsaI linearized vector (50 ng / μL) 5 μL (250 ng), double-stranded Oligo library (~10 ng / μL) 3 μL (~30 ng), 10× T4 DNA Ligase Buffer 5 μL, BsaI-HFv2 (20 U / μL) 1 μL, T4 DNA Ligase (400 U / μL) 1 μL, 10 mg / mL BSA 0.5 μL, Bring the volume to 50 μL. Cycling program: 37℃ for 5 min / 25℃ for 10 min, for a total of 40 cycles; final cycle: 50℃ for 10 min, 80℃ for 10 min.

[0179] ccdB reverse selection: Because the ccdB toxic protein is expressed lethally in competent cells, empty vectors integrating the ccdB gene cannot form colonies in E. coli; only clones that successfully replace ccdB (i.e., vectors with inserted sgRNA) can survive. Transform 2 μL of Golden Gate product into NEB 5-alpha (or DH5α, but must be ccdB-tolerant, such as DB3.1, common competent cells), plate on LB agar plates (15 cm in diameter) containing spectinomycin (50 mg / L), and incubate at 37°C for 16 h.

[0180] Library quality assessment criteria: colony count ≥ 100 times the number of sgRNA species; sgRNA insertion rate ≥ 85%.

[0181] 3.3 Determination of herbicide screening pressure

[0182] Imidazolium ethionine was added to the hairy root induction medium at concentrations of 0, 10, 15, 20, and 30 mg / L, with three biological replicates of 20 explants per replicate. After transformation of K599 Agrobacterium with a blank expression vector (without sgRNA), the explants were screened for 3 weeks on media containing different concentrations of imidazoline ethionine.

[0183] Hairy root induction rate and transgenic positivity rate were statistically analyzed (Bar gene PCR detection). Hairy root induction rate (%) = (number of explants producing hairy roots / total number of inoculated explants) × 100%; Transgenic positivity rate (%): Genomic DNA was extracted from each hairy root in each treatment group, and PCR detection was performed using Bar gene-specific primers. Bar primers: F 5'-GAAGTCCAGCTGCCAGAAAC-3' (SEQ ID NO.47); R 5'-GATCTCGGTGACGGGCAGGA-3' (SEQ ID NO.48), expected product 350 bp.

[0184] Positive rate = (Number of Bar gene PCR-positive hairy roots / Total number of hairy roots detected) × 100%. Criteria for determining the optimal selection pressure: Hairy root induction rate of 20-40%, a decrease of 50-70% compared to the control group, and a transgenic positive rate ≥ 60%.

[0185] 3.4 Screening for PsALS resistance sites in peas

[0186] The ABE-Lib and CBE-Lib library plasmids were transformed into K599 competent cells, respectively. After electroporation, K599 cells were plated onto eight 15 cm diameter YEB plates (containing 50 mg / L spectinomycin) and incubated at 28°C for 48 h. Bacterial growth was then scraped off using a spreader after adding 3 mL of YEB liquid medium, and the mixture was combined and the bacterial culture extracted. Adjust to =0.8.

[0187] Pea explants were infected with Agrobacterium library culture, co-cultured, and then transferred to a medium containing the optimal concentration of imidazoline. ≥200 explants were used for each library type, with 3 biological replicates, and selection cultured for 3-4 weeks.

[0188] Genomic DNA was extracted from resistant hairy roots that grew normally on a medium containing imidazole nicotinic acid. PCR amplification and Sanger sequencing were performed using universal primers targeting the sgRNA backbone region to obtain the sgRNA target sequence and determine the sgRNA number and target gene location carried by each resistant hairy root.

[0189] Hi-TOM high-throughput sequencing was used to identify the editing sites of the PsALS gene in each resistant hairy root.

[0190] Based on the sgRNA target sites identified above, Hi-TOM amplification primers were designed for the PsALS target fragments corresponding to each target gene (extending 200-400 bp from both sides of the sgRNA target site, with a product size of 400-700 bp).

[0191] Multiplex PCR strategy: Primers targeting the same gene region (e.g., PsALS1 Exon1-2) are designed with a specific tag sequence at the 3' end. Amplification and index addition are completed through two rounds of PCR. First round PCR (target-specific amplification): Reagents are the same as above. PCR program: 94°C for 2 min; 94°C for 30 sec, 62-65°C for 30 sec (depending on the primers). (Value optimization), 68°C for 40 seconds, 35 cycles; 68°C for 5 min. Second round PCR (library index addition): The first round PCR product was diluted 10-fold as a template, and PCR amplification was performed using universal index primers (containing Illumina P5 / P7 sequences and barcodes) (8 cycles) to construct the sequencing library.

[0192] Library quality assessment: Quantification was performed using an Agilent 2100 Bioanalyzer and Qubit. The library fragment size met expectations (400-700 bp), and the concentration was ≥10 nM. The library was sequenced using PE250 sequencing on an Illumina NovaSeq 6000 platform, with a target of ≥50,000 reads per sample.

[0193] Data analysis process:

[0194] ① Use Cutadapt to remove primer and adapter sequences (error rate ≤ 0.1);

[0195] ② Use BWA-MEM to align clean reads to the PsALS1 / 2 / 3 reference sequences;

[0196] ③ Use CRISPResso2 (v2.1.3) for quantitative analysis of editing events. Parameter settings: -q 30 (minimum base mass fraction), -w 20 (edit window size), --min_frequency_for_conversion 0.01;

[0197] ④ Output the editing efficiency, editing spectrum (editing frequency of each base position), and editing product type (homozygous / heterozygous, deletion, insertion) for each sample.

[0198] ⑤Focus on analyzing the editing events within the editing window that lead to amino acid substitutions, and identify at which amino acid sites the PsALS gene in each resistant hairy root was substituted.

[0199] Example 4: Creation of new herbicide-resistant pea germplasm

[0200] Technical routes for the creation of new herbicide-resistant pea germplasm, such as Figure 5 As shown.

[0201] 4.1 Construction of Multi-Target Dual-Single Base Editing Vectors

[0202] (1) Validation and editing strategy selection for key sites

[0203] Based on the Hi-TOM sequencing data of resistant hairy roots obtained in Example 3, all amino acid substitution sites leading to resistance were summarized. They were categorized according to the following rules: Case A—All key sites were generated by ABE (A>G) or all by CBE (C>T), and a tandem expression vector containing multiple sgRNAs was constructed using a single-base editor; Case B—Mixed editing, with key sites involving both ABE and CBE editing types, was performed using a dual single-base editor.

[0204] (2) Assembly of multiple sgRNA tandem expression cassettes

[0205] The tRNA-sgRNA modules are tandemly linked. Each sgRNA is added sequentially to the vector's sgRNA array using the Golden Gate method. Adjacent sgRNAs are separated by tRNA sequences. During RNA processing, the tRNAs are precisely cleaved by RNase P and RNase Z, releasing individual sgRNAs.

[0206] Construction of dual sgRNA vectors: Using a target vector (PsABE8e-tRNA-sgRNA1 or PsiA3A-tRNA-sgRNA1) already containing one tRNA-sgRNA as the recipient vector, linearize it again with BsaI, and then ligate the second tRNA-sgRNA2 downstream of the first sgRNA expression cassette via a Golden Gate. This process can be repeated to add a third and fourth sgRNA.

[0207] Multiple sgRNA tandem method: ① When designing a single sgRNA, adjacent sgRNAs are separated by a tRNA sequence (tRNA-sgRNA1-tRNA-sgRNA2...); ② During RNA processing, the tRNA is precisely cleaved by RNase P and RNase Z, releasing a single sgRNA and avoiding interference from long transcripts; ③ The multiple sgRNA array is directly cloned into the BsaI site of the vector in the form of PCR synthesis.

[0208] (3) Construction of the double-single base editor (A&C Twin-BE)

[0209] Construction of the dual-base editor (A&C Twin-BE): Two deaminases, TadA-8e and A3A, were simultaneously fused to the N-terminus of nCas9(D10A). Comparison of the two structures:

[0210] Structure I (N-terminal dual fusion): SV40 NLS - TadA-8e - - A3A - XTEN Linker - nCas9(D10A) - UGI + 3×free UGI - 3×SV40 NLS (Two deaminases are tandemly linked to the N-terminus of nCas9, with the middle terminus at...) Linker connection).

[0211] Structure II (N-terminal + C-terminal separation and fusion): SV40 NLS - TadA-8e - XTEN Linker - nCas9(D10A) - A3A - - UGI+3×free UGI - 3×SV40 NLS (TadA-8e at the N end, A3A at the C end, UGI after A3A).

[0212] The two structures were synthesized to obtain complete coding sequences, which were then cloned into the pHUC-PsU6.3 SpR sg2.0 vector backbone to construct a dual single-base editing intermediate vector.

[0213] Subsequently, multiple sgRNAs (designed for different key sites) were tandemly assembled into the sgRNA array of the dual single-base editing vector to obtain a multi-target dual single-base editing vector.

[0214] 4.2 Stable genetic transformation and creation of resistant germplasm

[0215] The targeted editing vector or the double single-base editing vector was transformed into Agrobacterium tumefaciens EHA105 competent cells, and then transformed into pea explants (Zhongwan 6) via Agrobacterium-mediated transformation.

[0216] Transformation procedure: Take 4-5 day old sterile seedlings and make a 3-5 mm longitudinal incision at the hypocotyl. Immerse the explants in EHA105 bacterial suspension ( =0.5, containing 100 μM AS), and after shaking and incubation for 30 min, transferred to co-culture medium (containing 100 μMAS, 1 mg / L 6-BA, and 0.1 mg / L NAA), and incubated in the dark at 25°C for 3 days.

[0217] After co-culturing, the samples were transferred to selection medium (containing 250 mg / L cefotaxime, 150 mg / L termethin, and 5-10 mg / L glufosinate) and cultured under 25°C light (16 h / 8 h, light intensity 50 μmol / m² / s), with the medium changed every 14 days. After 4-6 weeks of selection, adventitious shoots were cut off and transferred to adventitious shoot elongation medium (containing 0.5 mg / L 6-BA, 0.5 mg / L glufosinate, and termethin). 3-5 mg / L glufosinate. After 4-8 weeks of culture, the elongated adventitious shoots are transferred to rooting medium (containing 0.5 mg / L IBA and 0.2 mg / L NAA). After 2-4 weeks of culture, the seedlings are hardened off and transplanted.

[0218] 4.3 Molecular identification of T0 generation regenerated plants

[0219] Genomic DNA was extracted from leaves of T0 generation regenerated plants and subjected to the following tests:

[0220] (1) Transgenic positive identification: Dual PCR detection was performed using Bar gene and nptII gene specific primers. Dual PCR system: Bar-F / R and nptII-F / R primer pairs were mixed in the same PCR tube.

[0221] (2) Detection of Cas9 / nCas9 gene fragment: A 500 bp fragment of the nCas9 gene was amplified using specific primers.

[0222] (3) Target site editing detection: PCR amplification and Sanger sequencing were performed using target site-specific Hi-TOM primers. The editing status of each target site was analyzed (including editing efficiency, editing homozygosity, etc.).

[0223] (4) Off-target analysis: Cas-OFFinder is used to predict potential off-target sites (≤4 mismatches are allowed), and Sanger sequencing or targeted deep sequencing is performed for detection.

[0224] 4.4 Herbicide Resistance Identification

[0225] Once the T0 generation plants reached the 4-6 leaf stage, a herbicide spraying experiment was conducted. Five treatments were established: 0 (water control), 25 (0.5×), 50 (1×), 100 (2×), and 200 (4×) g ai / ha imidazoline. Resistance levels were assessed 21 days after spraying according to the following criteria:

[0226] Complete resistance (HR) Normal growth and development, no visible pesticide damage Level 0 High resistance (R) Some leaf edges are slightly yellowed (<10% of leaf area). Level 1 Moderate resistance (MR) Leaves turn yellow over 10-30% of their surface area, and growth is slightly inhibited. Level 2 Partially Sensitive (MS) Leaves are yellowed over 30-60% of their surface area, and stunting is evident. Level 3 Sensitive (S) Severe yellowing and necrosis, significantly stunted Level 4 Highly sensitive (HS) Plant death Level 5

[0227] 4.5 Identification and Validation of Key Resistance Sites

[0228] Genomic DNA was extracted from leaves of resistant plants (HR and R grades), and deep sequencing of all target sites was performed using the Hi-TOM method to precisely determine the mutation patterns of the PsALS family in each plant. The differences in resistance phenotypes between single-site mutant plants and multi-site combination mutant plants were analyzed, and the resistance levels and agronomic traits of different mutant site combinations were compared. Finally, the key amino acid site combinations conferring herbicide resistance (≥2 grade or higher resistance) were determined.

[0229] Positive T0 plants retaining good agronomic traits were self-pollinated, and T1 generation seeds were harvested. Continuous screening up to the T3 generation yielded genetically stable herbicide-resistant pea germplasm.

[0230] Example 5: Determination of editing parameters and verification of application effects of the pea base editing system

[0231] 5.1 Editing Efficiency Measurement

[0232] At the hairy root level, using the final optimized PsABE8e and PsiA3A systems, Hi-TOM sequencing analysis was performed on ≥30 independent hairy roots, targeting the PsALS1, PsALS2, and PsALS3 genes (3 sgRNA sites for each gene). Expected editing efficiencies: ABE system 20%-60%, CBE system 25%-70%, dual single-base editor A→G conversion efficiency 10%-40%, C→T conversion efficiency 15%-50%.

[0233] At the level of regenerated plants, Sanger sequencing of target sites was performed on T0 generation regenerated plants to statistically analyze the incidence of editing events.

[0234] 5.2 Edit-specificity assessment

[0235] Potential off-target sites for each sgRNA were predicted genome-wide using Cas-OFFinder (≤5 mismatches allowed). The top 10 potential off-target sites were sequenced using Hi-TOM deep sequencing (≥5,000 reads / site coverage) at the hairy root level, and the top 5 potential off-target sites were sequenced using Sanger sequencing at the T0 plant level.

[0236] 5.3 Resistance Identification of Herbicide-Resistant Germplasm

[0237] (1) Potted plant resistance identification

[0238] The obtained T2-T3 generation herbicide-resistant homozygous pea lines and wild-type Zhongwan 6 (control) were sown in flowerpots (20 cm in diameter, 3 plants per pot, 5 pots per line). When the plants reached the 4-6 leaf stage, different concentrations of imidazoline were sprayed. After 21 days of treatment, plant height (cm, length from stem base to growing point), aboveground fresh weight (g / plant), leaf SPAD value (measured using a SPAD-502 chlorophyll meter on the third unfolded leaf), and mortality rate (%) were measured as follows: number of dead plants / total number of treated plants × 100%.

[0239] (2) Herbicide cross-resistance spectrum test

[0240] Cross-resistance tests were conducted on the optimal resistant lines using different ALS inhibitor herbicides. The herbicide types and dosages included: imidazolinone (50, 100, 200 g ai / ha); chlorsulfuron (15, 30, 60 g ai / ha); bensulfuron (22.5, 45, 90 g ai / ha); and bispyribac-sodium (30, 60, 120 g ai / ha).

[0241] (3) Comprehensive evaluation of agronomic traits of resistant lines

[0242] Under non-herbicide treatment conditions, the main agronomic traits of herbicide-resistant lines and wild-type controls were compared: plant height (maturity), number of nodes on the main stem, height of the first branch; number of pods per plant, number of seeds per pod, weight of 100 seeds; plot yield (kg / plot); growth period (emergence-flowering, flowering-maturity days).

[0243] (4) Statistical analysis

[0244] All data were statistically analyzed using R (v4.1.0), employing one-way ANOVA and Tukey HSD multiple comparison test (α=0.05). GraphPad Prism 9.0 was used for plotting, with bar charts labeled as mean ± standard error (SEM). Different letters indicate statistical significance at the P<0.05 level.

[0245] (5) Experimental results

[0246] Editing efficiency analysis results

[0247] Hi-TOM was used to statistically analyze all editing events. Results showed that the PsABE8e system could stably achieve A→G editing; the PsiA3A system could stably achieve C→T editing; and there were some differences in editing efficiency among different target sites. However, overall, the ABE editing efficiency reached 2%–12%; and the CBE editing efficiency reached 0.5%–16%, indicating that the established system can meet the requirements for single-base editing in peas.

[0248] Edit-specific analysis results

[0249] Hi-TOM analysis was performed on the predicted off-target sites. No editing events were detected at the vast majority of off-target sites. Only a few sites showed extremely low-frequency editing, which did not affect the coding region. This indicates that the established base editing system has high editing accuracy.

[0250] Herbicide-resistant germplasm evaluation

[0251] Currently, a comprehensive evaluation of the obtained herbicide-resistant edited pea lines is underway, including pot resistance, cross-resistance to ALS inhibitor herbicides, and analysis of major agronomic traits. Through different herbicide treatments, plant growth, herbicide damage symptoms, survival rate, and related agronomic indicators are investigated. The herbicide resistance, cross-resistance, and overall agronomic performance of different edited lines are systematically evaluated, providing a basis for screening genetically stable, highly resistant, and agronomically beneficial new herbicide-resistant pea germplasm.

[0252] The pea single-base editing system and its application method provided by this invention can be widely used in pea functional genomics research, gene function identification of important traits, and genetic improvement. The herbicide-resistant pea germplasm created using this invention can be directly used in pea production, effectively solving the problem of weed control in pea fields, and has significant agricultural application value and industrialization prospects.

Claims

1. A single-base editing system suitable for peas, characterized in that, The system includes an adenine base editor and / or a cytosine base editor; The adenine base editor comprises a first fusion protein, which, from the N-terminus to the C-terminus, comprises: an SV40 nuclear localization signal, TadA-8e adenine deaminase, a first linker peptide, nCas9 (D10A) protein, and a 3×SV40 nuclear localization signal. The cytosine base editor comprises a second fusion protein, which, from the N-terminus to the C-terminus, comprises: an SV40 nuclear localization signal, an A3A cytosine deaminase, a second linker peptide, an nCas9 (D10A) protein, a uracil glycosylase inhibitor UGI, and a 3×SV40 nuclear localization signal. The amino acid sequence of the nCas9(D10A) protein is shown in SEQ ID NO.1, the amino acid sequence of the TadA-8e adenine deaminase is shown in SEQ ID NO.2, the amino acid sequence of the A3A cytosine deaminase is shown in SEQ ID NO.3, the sequence of the first linker peptide is shown in SEQ ID NO.5, the sequence of the second linker peptide is shown in SEQ ID NO.6, and the sequence of the uracil glycosylation inhibitor UGI is shown in SEQ ID NO.

4.

2. The single-base editing system for peas as described in claim 1, characterized in that, The coding sequences of the nCas9(D10A) protein, TadA-8e adenine deaminase, A3A cytosine deaminase, and uracil glycosylation inhibitor UGI were all optimized using pea codons. The optimization principle was to prioritize the use of codons with a synonymous codon frequency of ≥30% in peas, and to remove AT-rich regions, cryptic splice sites, and internal repetitive sequences.

3. The single-base editing system for peas as described in claim 1, characterized in that, The adenine base editor is driven by the enhanced promoter en35s; the single base editing system also includes an sgRNA expression cassette; the sgRNA expression cassette is transcribed by the pea endogenous U6 promoter PsU6.3, and the sgRNA expression cassette contains a tRNA-sgRNA fusion expression unit.

4. A double-single-base editor, characterized in that, The dual monobase editor can simultaneously perform A·T to G·C conversion and C·G to T·A conversion in the same cell.

5. The dual-single-base editor as described in claim 4, characterized in that, The dual monobase editor also includes a third fusion protein, which is selected from one of the following structures: Structure I: From the N-terminus to the C-terminus, it contains the SV40 nuclear localization signal, TadA-8e adenine deaminase, Flexible linker peptide, A3A cytosine deaminase, XTEN linker peptide, nCas9 (D10A) protein, uracil glycosylation inhibitor UGI, and 3×SV40 nuclear localization signal; Structure II: From the N-terminus to the C-terminus, it contains, sequentially, the SV40 nuclear localization signal, TadA-8e adenine deaminase, XTEN linker peptide, nCas9 (D10A) protein, and A3A cytosine deaminase. Flexible linker peptide, uracil glycosylation inhibitor UGI, and 3×SV40 nuclear localization signal.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the coding sequence of the single-base editing system according to any one of claims 1 to 3 or the double single-base editor according to claims 4 to 5.

7. The application of the single-base editing system according to any one of claims 1-3, the double single-base editor according to claim 4 or 5, or the recombinant expression vector according to claim 6 in pea gene editing.

8. A pea sgRNA library, characterized in that, The pea sgRNA library contains multiple sgRNAs targeting the pea PsALS gene family, including PsALS1, PsALS2, and PsALS3. The sgRNA library also contains eight sgRNAs targeting the PsALS1 gene, of which Targets 1-4 are for adenine base editing and Targets 5-8 are for cytosine base editing.

9. A method for screening herbicide resistance sites in the PsALS gene of peas, characterized in that, Includes the following steps: (1) Constructing an sgRNA library targeting the pea PsALS gene family; (2) The sgRNA library is cloned into the recombinant expression vector of claim 6 to obtain the sgRNA library plasmid; (3) The sgRNA library plasmid was transformed into Agrobacterium rhizogenes K599 to infect pea explants and induce the production of transgenic hairy roots; (4) The hairy roots were cultured on a screening medium containing an acetolactate synthase ALS inhibitor herbicide to screen for resistant hairy roots; (5) Genomic DNA was extracted from resistant hairy roots and the sgRNA target sequence and PsALS gene editing site were identified by sequencing; (6) Identify the key amino acid sites of the PsALS gene that confers herbicide resistance.

10. A method for creating herbicide-resistant pea germplasm, characterized in that, Includes the following steps: (1) Screening to obtain the key amino acid sites of the PsALS gene that confers herbicide resistance; (2) Design and synthesize the corresponding sgRNA based on the key amino acid sites; (3) One or more sgRNAs are assembled into the recombinant expression vector of claim 6 to obtain a targeted editing vector; when multiple sgRNAs are assembled, each sgRNA is separated by a tRNA sequence to form a tRNA-sgRNA tandem expression cassette; (4) When the key amino acid site requires both A→G and C→T editing types, multiple sgRNAs are assembled into the double single base editor of claim 4 to obtain a multi-target double single base editing vector. (5) The targeted editing vector or the double single-base editing vector was transformed into Agrobacterium tumefaciens EHA105, and pea explants were transformed by Agrobacterium-mediated transformation. (6) Screen positive transformants on a culture medium containing screening agents, induce adventitious bud differentiation, elongation and rooting, and obtain T0 generation regenerated plants; (7) Molecular identification and herbicide resistance identification were performed on the T0 generation regenerated plants, and herbicide-resistant positive plants were screened; (8) Self-pollinate positive plants to obtain genetically stable herbicide-resistant pea germplasm.

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