Pilot editing system and application thereof

Abstract

The invention discloses a pilot editing system. The pilot editing system comprises xseA4crRNA or a coding gene of the xseA4crRNA, sbcB3crRNA or a coding gene of the sbcB3crRNA, sbcB6crRNA or a coding gene of the sbcB6crRNA and exoX4crRNA or a coding gene of the exoX4crRNA, and / or PE protein or a coding gene thereof; and / or, epegRNA or a coding gene thereof. According to the pilot editing system provided by the invention, sbcB, xseA and exoX are knocked out at the same time, so that the efficiency of point mutation can be improved by 3.6-42.3 times, and the efficiency of point mutation at multiple sites is improved from 13.9% to 64.5%; the sbcB, the xseA and the exoX are knocked out at the same time, so that the insertion efficiency and the deletion efficiency can be improved by 6.6-99.5 times and 23.1-69.6 times respectively, and the insertion efficiency and the deletion efficiency at multiple sites are improved from 2.1% to 20.6% and from 0.5% to 26.8% respectively.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a lead editing system and its application. Background Art

[0002] Gene editing is an important tool for studying the function of genes in model organisms. Since its inception, the CRISPR / Cas (Clustered regularly interspaced short palindromic repeats / CRISPR-associated protein) genome editing system has been widely used in biology, medicine, agronomy and other fields due to its simplicity and efficiency. By fusing inactivated Cas nucleases with base deaminases or reverse transcriptases, the CRISPR / Cas system can be further modified into base editors (BE) and prime editors (PE). Prime editors can achieve insertions, deletions and all types of point mutations at targeted locations without generating double-strand DNA breaks, and have been widely used in multiple cell lines such as fruit flies, zebrafish, wheat, rice, mouse embryos and human stem cells.

[0003] The prime editing system consists of pegRNA (Prime editing guide RNA) and Cas9nickase fused with reverse transcriptase. During the prime editing process, the PE-pegRNA complex binds to the targeted chain and creates a nick on the non-targeted chain. The released 3'DNA can bind to the PBS (Primer bonding site) in the pegRNA and serve as a primer for reverse transcription. Under the action of reverse transcriptase, the sequence in the reverse transcription template in the pegRNA is reverse transcribed to form a 3'DNA flap containing the editing sequence, which is then integrated into the genome during the subsequent DNA repair process, so that the editing results are permanently preserved.

[0004] Prime editing was initially developed in human cells and quickly applied to multiple species. However, the efficiency of prime editing systems varies significantly across species and within the same species. Therefore, there is an urgent need to develop methods that can universally improve prime editing efficiency. Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a lead editing system and application thereof, so as to solve the problems in the prior art.

[0006] The present application provides a CRISPRi expression construct, which includes an xseA4 crRNA nucleotide sequence, a sbcB3 crRNA nucleotide sequence, a sbcB6 crRNA nucleotide sequence, and an exoX4 crRNA nucleotide sequence.

[0007] This application also provides the use of the above-mentioned CRISPRi expression construct in gene editing.

[0008] The present application also provides an epegRNA, which includes a pegRNA spacer, a pegRNA 3'extension and a pegRNA scaffold.

[0009] The present application also provides an epegRNA expression construct, which contains the epegRNA as described in claim 4 or 5.

[0010] The present application also provides a lead editing system, including: xseA4 crRNA or its encoding gene, sbcB3 crRNA or its encoding gene, sbcB6 crRNA or its encoding gene, and exoX4 crRNA or its encoding gene; PE protein or its encoding gene; epegRNA or its encoding gene.

[0011] The present application also provides a lead editing method, comprising contacting the above-mentioned lead editing system with a target gene for gene editing.

[0012] The present application also provides an engineered bacterium comprising the above-mentioned CRISPRi expression construct.

[0013] The present application also provides an engineered bacterium, which is obtained by gene editing the engineered bacterium using the above-mentioned lead editing method.

[0014] The present application also provides an engineering bacteria construction kit, comprising the above-mentioned lead editing system.

[0015] The present application also provides the application of the above-mentioned lead editing system or the above-mentioned lead editing method in gene editing.

[0016] The beneficial effects of the embodiments of the present application include but are not limited to: (1) The present application provides a fusion protein having gene editing function and a novel and efficient lead editing system containing the fusion protein, which is characterized by inhibiting the hydrolysis of the 3'DNA produced by the lead editing. The invention relates to a key 3'→5' single-stranded DNA exonuclease of flap, which improves the efficiency of lead editing including point mutation, insertion and deletion; (2) the fusion protein provided in the present application and the lead editing system containing it are used to achieve at least one of the correction of pathogenic sites, gene function research, enhancement of cell function, and cell therapy; (3) the lead editing system provided in the present application can achieve efficient lead editing in bacteria: simultaneous knockout of sbcB, xseA and exoX can increase the efficiency of point mutation by 3.6 to 42.3 times, and achieve an increase in the efficiency of point mutation at multiple sites from 13.9% to 64.5%; simultaneous knockout of sbcB, xseA and exoX can increase the efficiency of insertion or deletion by 6.6 to 99.5 times and 23.1 to 69.6 times, respectively, and increase the efficiency of insertion or deletion at multiple sites from 2.1% to 20.6% and from 0.5% to 26.8%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present application will be further described in terms of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are non-limiting, and include:

[0018] Figure 1 Schematic diagram of the BacPE prime editing system for wild-type Escherichia coli. Prior to prime editing, CRISPRi was used to inhibit the transcription of the key genes impairing prime editing, sbcB, xseA, and exoX. The PE2 protein and epegRNA were encoded on two plasmids, respectively.

[0019] Figure 2 To construct the CRISPRi inhibition system plasmid, four crRNAs (CRISPR RNAs), xseA4, sbcB3, sbcB6, and exoX4, were inserted into the plasmid pBbS8c-ddcpf1 (SEQ ID NO: 6) using Golden Gate assembly.

[0020] Figure 3 The figure shows the transcriptional levels of genes involved in suppressing prime editing by CRISPRi. As shown in the figure, CRISPRi significantly suppressed the transcriptional levels of these genes.

[0021] Figure 4Construction of a PE protein expression plasmid: The p15a replicon sequence was amplified from the pET28a (SEQ ID NO: 19) plasmid, the CbR resistance gene was amplified from the pKD46_Cas9 (SEQ ID NO: 20) plasmid, and the SpCas9_M-MLVmut gene was amplified using the SpCas9_M-MLVmut gene sequence as a template. The resulting two DNA fragments were assembled using Gibson ligation.

[0022] Figure 5 The pegRNA spacer, pegRNA 3'extension, and pegRNA scaffold were inserted into the pegRNA vector empty plasmid pegRNA2_tevopreQ1 (SEQ ID NO: 12) by Golden Gate ligation: the plasmid pegRNA2_tevopreQ1 was first digested overnight with BsaI, and then the linearized plasmid was purified using the TIANquick Midi Purification Kit; then, the purchased insert was annealed and phosphorylated according to the instructions of T4 PNK, and then the four fragments were assembled together using Golden Gate ligation.

[0023] Figure 6 To construct and culture Escherichia coli MG1655-i containing pBbS8c-ddCpf1-Δ plasmid, SpPE plasmid and epegRNA plasmid, and to verify the editing efficiency of the BacPE prime editing system.

[0024] Figure 7 The editing efficiency of BacPE for different types of editing was characterized. (A) Substitution editing in xylB of E. coli MG1655 using BacPE. The epegRNA cut site is marked with a black triangle; (B) Insertion editing in xylB of E. coli MG1655 using BacPE; (C) Deletion editing in xylB of E. coli MG1655 using BacPE. As shown in the figure, the efficiency of prime editing is high when point mutations occur at positions +5 and +6; when inserting at position +6, the efficiency of prime editing is high; when performing deletion editing, the efficiency of deletion is high between positions +1 and +7.

[0025] Figure 8 Figure 2 shows the effects of sbcB, xseA, and exoX on prime editing efficiency in Klebsiella pneumoniae. As shown in the figure, Klebsiella pneumoniae exhibits DNA repair mechanisms similar to those in Escherichia coli for prime editing intermediates. DETAILED DESCRIPTION

[0026] To more clearly illustrate the technical solutions of the embodiments of this specification, the following briefly describes the drawings required for describing the embodiments. Obviously, the drawings described below are merely examples or embodiments of this specification. Those skilled in the art can apply this specification to other similar scenarios based on these drawings without inventive effort. Unless otherwise apparent from the context or otherwise noted, the same reference numerals in the figures represent the same structure or operation.

[0027] As used in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not refer to the singular but also include the plural. Generally speaking, the terms "comprises" and "include" only indicate the inclusion of the steps and elements specifically identified, and these steps and elements do not constitute an exclusive list. A method or apparatus may also include other steps or elements.

[0028] Flowcharts are used throughout this specification to illustrate the operations performed by systems according to embodiments of this specification. It should be understood that preceding or following operations do not necessarily need to be performed in exact order. Instead, the steps may be processed in reverse order or simultaneously. Furthermore, other operations may be added to these processes, or one or more operations may be removed from these processes.

[0029] Because the editing efficiency of prime editing systems varies significantly across species and across sites within the same species, there is currently no prime editing system that can efficiently edit bacterial genomes. The applicant's prime editing system can inhibit the transcription of key bacteria genes, sbcB, xseA, and exoX, through CRISPRi, thereby improving the efficiency of prime editing for point mutations, insertions, and deletions.

[0030] The present application provides a CRISPRi expression construct, which includes an xseA4 crRNA nucleotide sequence, a sbcB3 crRNA nucleotide sequence, a sbcB6 crRNA nucleotide sequence, and an exoX4 crRNA nucleotide sequence.

[0031] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0032] "Construct" herein refers to a polynucleotide that can carry at least one polynucleotide fragment. The construct can deliver a fragment of nucleic acid, each polynucleotide, into a host cell. It can include at least one expression cassette comprising a regulatory sequence for correctly expressing the polynucleotide incorporated therein. The polynucleotide to be introduced into the cell (for example, a polynucleotide encoding a product of interest or a selective marker) can be inserted into the expression cassette of a vector for expression therefrom. Constructs according to the present application can exist in a circular or linear (linearized) form, and also include expression construct fragments. The term "construct" also includes artificial chromosomes or similar individual polynucleotides that allow the transfer of exogenous nucleic acid fragments.

[0033] The "sequence" herein should generally be understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the amino acid sequence, unless a more limited interpretation is required herein.

[0034] As used herein, the terms "polynucleotide," "nucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably to refer to nucleic acids including DNA, RNA, derivatives thereof, or combinations thereof.

[0035] In some embodiments, the xseA4 crRNA nucleotide sequence may be as shown in SEQ ID NO. 15. In some embodiments, the sbcB3 crRNA nucleotide sequence may be as shown in SEQ ID NO. 16. In some embodiments, the sbcB6 crRNA nucleotide sequence may be as shown in SEQ ID NO. 17. In some embodiments, the exoX4 crRNA nucleotide sequence may be as shown in SEQ ID NO. 18.

[0036] In some embodiments, the nucleotide sequence of the CRISPRi expression construct may be as shown in SEQ ID NO.1.

[0037] This application also provides the use of the above-mentioned CRISPRi expression construct in gene editing.

[0038] In some embodiments, the gene editing can be selected from at least one of gene cleavage, gene deletion, gene insertion, base substitution, point mutation, transcription inhibition, transcription activation, or base editing. In some embodiments, preferably, the gene editing can be selected from gene deletion, gene insertion, or point mutation.

[0039] The present application also provides an epegRNA, which includes a pegRNA spacer, a pegRNA 3'extension and a pegRNA scaffold.

[0040] In some embodiments, the nucleotide sequence of the epegRNA is selected from a sequence shown in any one of SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO.11.

[0041] The present application also provides an epegRNA expression construct, which may contain the above-mentioned epegRNA.

[0042] In some embodiments, the epegRNA expression construct can be obtained by inserting the above-mentioned epegRNA into an empty plasmid pegRNA2_tevopreQ1. In some embodiments, the nucleotide sequence of the pegRNA2_tevopreQ1 can be as shown in SEQ ID NO.12.

[0043] In some embodiments, the nucleotide sequence of the epegRNA expression construct can be selected from the sequence shown in any one of SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7.

[0044] The term "gRNA" refers to an RNA molecule capable of directing a CRISPR effector with nuclease activity to target a specified target nucleic acid.

[0045] The present application also provides a lead editing system, including: xseA4 crRNA or its encoding gene, sbcB3 crRNA or its encoding gene, sbcB6 crRNA or its encoding gene, and exoX4 crRNA or its encoding gene; and / or, PE protein or its encoding gene; and / or, epegRNA or its encoding gene.

[0046] In some embodiments, the coding sequence of the xseA4 crRNA may be SEQ ID NO. 15. In some embodiments, the coding sequence of the sbcB3 crRNA may be SEQ ID NO. 16. In some embodiments, the coding sequence of the sbcB6 crRNA may be SEQ ID NO. 17. In some embodiments, the coding sequence of the exoX4 crRNA may be SEQ ID NO. 18. In some embodiments, the amino acid sequence of the PE protein may be as shown in SEQ ID NO. 3.

[0047] In some embodiments, the epegRNA may include a pegRNA spacer, a pegRNA 3'extension, and a pegRNA scaffold.

[0048] In some embodiments, the coding sequence of the epegRNA can be selected from the sequence shown in any one of SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO.11.

[0049] In some embodiments, the encoding nucleotide sequence of the PE protein may be as shown in SEQ ID NO.4.

[0050] In some embodiments, the lead editing system may include a construct. In some embodiments, the construct may include: the xseA4 crRNA or its encoding gene, sbcB3 crRNA or its encoding gene, sbcB6 crRNA or its encoding gene, and exoX4 crRNA or its encoding gene; and / or, the PE protein or its encoding gene; and / or, the epegRNA or its encoding gene.

[0051] In some embodiments, the construct includes: the above-mentioned CRISPRi expression construct; and / or, the PE protein expression construct; and / or, the above-mentioned epegRNA expression construct.

[0052] In some embodiments, the nucleotide sequence of the PE protein expression construct may be as shown in SEQ ID NO. 2. In some embodiments, the molar ratio of the PE protein expression construct to the epegRNA expression construct may be 1:1.

[0053] In some embodiments, the PE protein expression construct may include a Cas9 protein framework element, a replicon element, and a resistance gene expression element. In some embodiments, preferably, the PE protein expression construct may include a Cas9 protein framework element as shown in SEQ ID NO.31, a P15a replicon element as shown in SEQ ID NO.30, and a resistance gene expression element as shown in SEQ ID NO.29.

[0054] In some embodiments, the Cas9 protein can be a Cas9 nuclease. In some embodiments, the Cas9 nuclease can be a Cas9 nuclease conventional in the art. In some embodiments, preferably, the Cas9 nuclease can be Syntrophomonas palmitatica Cas9 (SpCas9).

[0055] In some embodiments, the amino acid sequence of the PE protein may be as shown in SEQ ID NO. 3. In some embodiments, the coding sequence of the PE protein may be as shown in SEQ ID NO. 4.

[0056] In some embodiments, the nucleotide sequence of the PE protein expression construct may be as shown in SEQ ID NO.2.

[0057] The present application provides a lead editing method, comprising transferring the above-mentioned lead editing system into cells containing a target sequence for gene editing.

[0058] In some embodiments, the target sequence may be a DNA fragment of 20 bp in length following the PAM (Protospacer adjacent motif) sequence NGG. In some embodiments, preferably, the target sequence may be as shown in SEQ ID NO. 8 in the sequence listing.

[0059] SEQ ID NO. 8: 5'-ccccacgctttcgcaacttc-3'.

[0060] In some embodiments, the efficiency of lead editing can be improved by inhibiting or knocking out the expression of sbcB, exoX, and xseA 3'→5' single-stranded DNA exonucleases.

[0061] "Knockout" refers to the loss of expression of a specific protein in a cell. Knockout can be measured by detecting the amount of protein secreted from a tissue or cell population or by detecting the total cellular amount of protein in a tissue or cell population. In some embodiments, knockout can be the complete loss of expression of the target protein in a cell.

[0062] In some embodiments, the gene editing can be selected from at least one of gene cleavage, gene deletion, gene insertion, base substitution, point mutation, transcription inhibition, transcription activation, or base editing. In some embodiments, preferably, the gene editing can be selected from gene deletion, gene insertion, or point mutation.

[0063] In some embodiments, the method can be performed in vitro. In some embodiments, preferably, the method can be performed in cultured cells. In some embodiments, the method can be performed in vivo; preferably, the method can be performed in a mammal. In some embodiments, further preferably, the method can be performed in rodents or primates. In some embodiments, even further preferably, the method can be performed in mice or humans.

[0064] The present application also provides an engineered bacterium comprising the aforementioned CRISPRi expression construct. In some embodiments, the engineered bacterium can be a prokaryotic microorganism. In some embodiments, preferably, the engineered bacterium can be selected from any one of Actinomycetes, Bacillus, Streptomyces, Saccharomyces, Klebsiella pneumoniae, or Escherichia coli; in some embodiments, more preferably, the engineered bacterium can be selected from Klebsiella pneumoniae or Escherichia coli.

[0065] The present application also provides an engineered bacterium obtained by gene editing the engineered bacterium using the above-mentioned lead editing method. In some embodiments, the engineered bacterium can be a prokaryotic microorganism. In some embodiments, preferably, the engineered bacterium can be selected from any one of actinomycetes, Bacillus, Streptomyces, yeast, Klebsiella pneumoniae, or Escherichia coli; in some embodiments, more preferably, the engineered bacterium can be selected from Klebsiella pneumoniae or Escherichia coli.

[0066] The present application also provides an engineering bacteria construction kit, comprising the above-mentioned lead editing system.

[0067] The term "kit" refers to a packaged collection of related components, such as one or more polynucleotides or compositions, and one or more related materials, such as a delivery device (e.g., a syringe), solvents, solutions, buffers, instructions, or desiccant.

[0068] The present application also provides the application of the above-mentioned lead editing system or the above-mentioned lead editing method in gene editing.

[0069] In some embodiments, the gene editing can be selected from at least one of gene cleavage, gene deletion, gene insertion, base substitution, point mutation, transcription inhibition, transcription activation, or base editing. In some embodiments, preferably, the gene editing can be selected from gene deletion, gene insertion, or point mutation.

[0070] In some embodiments, the gene editing can be used to achieve at least one of correction of pathogenic sites, gene function research, enhancement of cell function, and cell therapy.

[0071] In an embodiment of the present application, a novel lead editing method based on CRISPR / SpCas9 nuclease is provided. The BacPE system consists of two parts: PE protein and epegRNA. The PE protein is formed by the fusion of Cas9 nickase and reverse transcriptase, and the epegRNA is composed of sgRNA, an RNA template for reverse transcription, and an RNA structural unit at the 3' end. Through the guiding and positioning functions of epegRNA, the PE-epegRNA complex binds to the target chain and produces a nick on the non-target chain. The freed 3'DNA can bind to the PBS in the epegRNA and serve as a primer for reverse transcription. Under the action of the reverse transcriptase, the sequence in the reverse transcription template in the epegRNA is reverse transcribed to form a 3'DNA flap containing the editing sequence, which is integrated into the genome during the subsequent DNA repair and replication process, and the editing results are permanently preserved.

[0072] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies. The quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0073] The sources of biological materials used in each example are as follows:

[0074] Isopropyl β-D-thiogalactopyranoside (IPTG), arabinose, glycerol, kanamycin (Km), carbenicillin (Cb), chloramphenicol (Cm), abramycin sulfate (Apr), and a column-based bacterial genomic DNA extraction kit were purchased from Sangon, China. The PCR enzymes Phanta and ChamQ Universal SYBR qPCR Master Mix were purchased from Novozymes, China. The DNA purification kits TIANquick Midi Purification Kit and TIANprep Mini Plasmid Kit were purchased from TIANGEN, China. The enzymes used in the experiment: T4 DNA ligase, Esp3I, BsaI, Gibson Assembly Master Mix, and DNase I were purchased from New Brunswick, New Brunswick, USA. Terrific Broth (TB) broth was purchased from MDbio, China. The RNA extraction kit TaKaRa MiniBEST Universal RNA Extraction Kit was purchased from Takara, Japan. Lysis Matrix B was purchased from MP Biosciences, USA. One-Step gDNA Removal and cDNA Synthesis SuperMix was purchased from Quanshijin Company, China.

[0075] The instruments used in this experiment are:

[0076] Gradient PCR instrument (Bio-Rad, USA), gel imager (Bio-Rad, USA), ultra-micro-spectrophotometer (Thermo, USA), electroporation system (Bio-Rad, USA), multifunctional microplate reader (Molecular Devices, USA), constant temperature shaking incubator (Minquan, China), desktop centrifuge (Thermo, USA), electronic balance (METTLER TOLEDO, Switzerland), FastPrep (MP, USA), qPCR instrument (Roche, USA), desktop refrigerated centrifuge (Hitachi, Japan).

[0077] Culture conditions of strains:

[0078] Escherichia coli (E. coli) TOP10 used for plasmid construction was cultured in TB medium at 37°C. E. coli MG1655 was cultured in TB medium at 37°C. K. pneumoniae 1.6366 was cultured in TB medium at 37°C. Antibiotic and chemical concentrations used were: Km, Cb, Cm, and Apr in E. coli and K. pneumoniae at 50 μg mL⁻¹; arabinose at 10 mM; and IPTG at 0.2 mM.

[0079] Example 1—CRISPRi inhibits the transcriptional levels of key DNA repair genes xseA, sbcB, or exoX

[0080] (1.1) Construction of the CRISPRi inhibition system pBbS8c-ddcpf1-Δ

[0081] The sequence of pBbS8c-ddcpf1-Δ is SEQ ID NO. 1. The specific construction method is as follows:

[0082] SEQ ID NO.1:

[0083] 5'-tagtccatgtgaccttcaccctcatggaccgcaaaaaaccccgcttcggcggggttttttcgctgcgcaactttggtaacttgatgtctacacctaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtgagcggataacaatttcaatttctactcttgtagatacgcaaccagcttccggtcactgtttttttgaagcttgggcccgaacaaaaactcggatccaaactcgagtaaggatctccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatacctagggtacgggttttgctgcccgcaaacgggctgttctggtgttgctagtttgttatcagaatcgcagatccggtagtccatgtgaccttcaccctcatggaccgcaaaaaaccccgcttcggcggggttttttcgctgcgcaactttggtaacttgatgtctacacctaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtgagcggataacaatttcaatttctactcttgtagatacctaatgatgaatgacggtaagtttttttgaagcttgggcccgaacaaaaactcggatccaaactcgagtaaggatctccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatacctagggtacgggttttg

[0084] ctgcccgcaaacgggctgttctggtgttgtagtccatgtgaccttcaccctcatggaccgcaaaaaaccccgcttcggcggggttttttcgctgcg

[0085] caactttggtaacttgatgtctacacctaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtgagcggataacaatttcaatt

[0086] tctactcttgtagatacgattacgaaacctttggcacgtttttttgaagcttgggcccgaacaaaaactcggatccaaactcgagtaaggatctccag

[0087] gcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcac

[0088] cttcgggtgggcctttctgcgtttatacctagggtacgggttttgctgcccgcaaacgggctgttctggtgttggctgaaagcgctatttcttccaga

[0089] attgccatgattttttccccacgggaggcgtcactggctcccgtgttgtcggcagctttgacctagtccatgtgaccttcaccctcatggaccgcaa

[0090] aaaaccccgcttcggcggggttttttcgctgcgcaactttggtaacttgatgtctacacctaaatgagctgttgacaattaatcatccggctcgtataa

[0091] tgtgtggaattgtgagcggataacaatttcaatttctactcttgtagattggcctgggatcaagtacagcaatttttttgaagcttgggcccgaacaaa

[0092] aactcggatccaaactcgagtaaggatctccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcg

[0093] gtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatacctagggtacgggttttgctgcccgcaaacgggctg

[0094] ttctggtgttctcgcaggctgtctatgtgtgactgttgagctgtaacaagttgtctcaggtgttcaatttcatgttctagttgctttgttttactggtttcacc

[0095] tgttctattaggtgttacatgctgttcatctgttacattgtcgatctgttcatggtgaacagctttgaatgcaccaaaaactcgtaaaagctctgatgtat

[0096] ctatcttttttacaccgttttcatctgtgcatatggacagttttccctttgatatgtaacggtgaacagttgttctacttttgtttgttagtcttgatgcttcact

[0097] gatagatacaagagccataagaacctcagatccttccgtatttagccagtatgttctctagtgtggttcgttgtttttgcgtgagccatgagaacgaa

[0098] ccattgagatcatacttactttgcatgtcactcaaaaattttgcctcaaaactggtgagctgaatttttgcagttaaagcatcgtgtagtgtttttcttagt

[0099] ccgttatgtaggtaggaatctgatgtaatggttgttggtattttgtcaccattcatttttatctggttgttctcaagttcggttacgagatccatttgtctatc

[0100] tagttcaacttggaaaatcaacgtatcagtcgggcggcctcgcttatcaaccaccaatttcatattgctgtaagtgtttaaatctttacttattggtttca

[0101] aaacccattggttaagccttttaaactcatggtagttattttcaagcattaacatgaacttaaattcatcaaggctaatctctatatttgccttgtgagtttt

[0102] cttttgtgttagttcttttaataaccactcataaatcctcatagagtatttgttttcaaaagacttaacatgttccagattatattttatgaatttttttaactgg

[0103] aaaagataaggcaatatctcttcactaaaaactaattctaatttttcgcttgagaacttggcatagtttgtccactggaaaatctcaaagcctttaacca

[0104] aaggattcctgatttccacagttctcgtcatcagctctctggttgctttagctaatacaccataagcattttccctactgatgttcatcatctgagcgtatt

[0105] ggttataagtgaacgataccgtccgttctttccttgtagggttttcaatcgtggggttgagtagtgccacacagcataaaattagcttggtttcatgct

[0106] ccgttaagtcatagcgactaatcgctagttcatttgctttgaaaacaactaattcagacatacatctcaattggtctaggtgattttaatcactataccaa

[0107] ttgagatgggctagtcaatgataattactagtccttttcccgggtgatctgggtatctgtaaattctgctagacctttgctggaaaacttgtaaattctg

[0108] ctagaccctctgtaaattccgctagacctttgtgtgttttttttgtttatattcaagtggttataatttatagaataaagaaagaataaaaaaagataaaaa

[0109] gaatagatcccagccctgtgtataactcactactttagtcagttccgcagtattacaaaaggatgtcgcaaacgctgtttgctcctctacaaaacaga

[0110] ccttaaaaccctaaaggcttaagtagcaccctcgcaagctcgggcaaatcgctgaatattccttttgtctccgaccatcaggcacctgagtcgctgt

[0111] ctttttcgtgacattcagttcgctgcgctcacggctctggcagtgaatgggggtaaatggcactacaggcgccttttatggattcatgcaaggaaac

[0112] tacccataatacaagaaaagcccgtcacgggcttctcagggcgttttatggcgggtctgctatgtggtgctatctgactttttgctgttcagcagttc

[0113] ctgccctctgattttccagtctgaccacttcggattatcccgtgacaggtcattcagactggctaatgcacccagtaaggcagcggtatcatcaaca

[0114] ggcttacccgtcttactgtccctagtgcttggattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttct

[0115] gaggtcattactggatctatcaacaggagtccaagcgagctcgatatcaaattacgccccgccctgccactcatcgcagtactgttgtaattcatta

[0116] agcattctgccgacatggaagccatcacaaacggcatgatgaacctgaatcgccagcggcatcagcaccttgtcgccttgcgtataatatttgcc

[0117] catggtgaaaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattggctgagacgaaaaa

[0118] catattctcaataaaccctttagggaaataggccaggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcg

[0119] tggtattcactccagagcgatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctt

[0120] tcattgccatacgaaattccggatgagcattcatcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacggtcttta

[0121] aaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactgactgaaatgcctcaaaatgttctttacgatgccattgggatat

[0122] atcaacggtggtatatccagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttc

[0123] attatggtgaaagttggaacctcttacgtgccgatcaacgtctcattttcgccagatatcgacgtcttatgacaacttgacggctacatcattcactttt

[0124] tcttcacaaccggcacggaactcgctcgggctggccccggtgcattttttaaatacccgcgagaaatagagttgatcgtcaaaaccaacattgcg

[0125] accgacggtggcgataggcatccgggtggtgctcaaaagcagcttcgcctggctgatacgttggtcctcgcgccagcttaagacgctaatccct

[0126] aactgctggcggaaaagatgtgacagacgcgacggcgacaagcaaacatgctgtgcgacgctggcgatatcaaaattgctgtctgccaggtg

[0127] atcgctgatgtactgacaagcctcgcgtacccgattatccatcggtggatggagcgactcgttaatcgcttccatgcgccgcagtaacaattgctc

[0128] aagcagatttatcgccagcagctccgaatagcgcccttccccttgcccggcgttaatgatttgcccaaacaggtcgctgaaatgcggctggtgcg

[0129] cttcatccgggcgaaagaaccccgtattggcaaatattgacggccagttaagccattcatgccagtaggcgcgcggacgaaagtaaacccact

[0130] ggtgataccattcgcgagcctccggatgacgaccgtagtgatgaatctctcctggcgggaacagcaaaatatcacccggtcggcaaacaaattc

[0131] tcgtccctgatttttcaccaccccctgaccgcgaatggtgagattgagaatataacctttcattcccagcggtcggtcgataaaaaaatcgagataa

[0132] ccgttggcctcaatcggcgttaaacccgccaccagatgggcattaaacgagtatcccggcagcaggggatcattttgcgcttcagccatacttttc

[0133] atactcccgccattcagagaagaaaccaattgtccatattgcatcagacattgccgtcactgcgtcttttactggctcttctcgctaaccaaaccggt

[0134] aaccccgcttattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgcgtaacaaaagtgtctataatcacggcagaaaagtc

[0135] cacattgattatttgcacggcgtcacactttgctatgccatagcatttttatccataagattagcggattctacctgacgctttttatcgcaactctctact

[0136] gtttctccatacccgtttttttgggaattcaaaagatctacaccgtcataaaatgcctcgcatgtcagtgttattctcttgcgcggttcataatccatgaa

[0137] actgtttttatgtaggaggtcgtgatgacccagtttgaaggttttaccaatctttatcaggtaagcaagacgctgagatttgagctgattccccaggg

[0138] caaaacactgaaacacattcaagagcaaggcttcattgaagaggataaggcccgaaacgaccattataaagaacttaaaccgattatcgatcgc

[0139] atctacaagacgtatgcagaccagtgtctgcaactggtgcagttagattgggaaaatttaagcgcggctatagactcttaccgtaaggagaaaac

[0140] agaagaaactcgtaatgcattaatcgaggagcaggctacataccgcaatgcaattcatgactattttattggtaggacagataatctaacggacgc

[0141] catcaacaagcgtcatgccgaaatatacaaaggtctgtttaaagcggagctgttcaacggtaaagtattaaagcagttaggtaccgtaacgacca

[0142] cggaacatgaaaatgccttgctgcgttcttttgataaattcactacgtatttttccgggttctacgagaatcgcaagaacgtgtttagcgcagaagat

[0143] attagtacggccattccgcatcgtattgttcaagacaattttccgaaatttaaagaaaattgtcatatttttactcgtctgatcacagcagtgccatcact

[0144] gcgagaacattttgaaaatgttaagaaggcgattggcattttcgtgtcaaccagcattgaagaagtgttttcattcccgttctataatcagcttcttacc

[0145] cagacacagattgacctatataatcagctcttaggtggtattagccgagaagctggaacggaaaagataaaaggcctgaatgaggtgctgaacc

[0146] tggcaattcaaaagaacgatgaaacagcgcacattattgcatctctcccccatcgatttataccgctctttaaacaaattctgtccgaccgcaacac

[0147] actgagctttatcctggaggaatttaaaagcgatgaagaagtaattcagagcttctgcaaatataaaacgttactgcgcaatgaaaacgtcctgga

[0148] aacggctgaggccctctttaacgaactgaacagcattgatctcacccatatttttatttcacataaaaagctggaaactatcagcagcgcgctgtgc

[0149] gaccattgggacacgctgagaaatgccctgtatgaacgtcgaatttccgagctgaccggaaagatcaccaagtcagctaaagagaaagtccag

[0150] cgctccctcaaacatgaagatattaacttacaagagattatttcggcggccggtaaggaactgtcggaggctttcaaacagaagacaagcgaaat

[0151] cctctcacatgcgcacgcggccctagatcagcctctgccgacgacgctgaaaaagcaggaagagaaagaaatcctgaaatctcaattagattct

[0152] ctactgggactgtaccacttgctggactggtttgcggtggacgagtctaatgaggtggaccctgaatttagcgcacgcctgaccggtataaaattg

[0153] gagatggaaccgtcgttgagcttttacaacaaagcacgcaactatgcgaccaaaaagccttattcagtggaaaaattcaaattgaacttccaaatg

[0154] ccgaccctagcgtcaggttgggatgtcaacaaagagaagaacaacggtgcgatcttatttgtgaaaaacggcctgtattatctgggcattatgcc

[0155] gaagcagaaaggccgttacaaggcactgtcattcgaaccgacggagaagaccagtgaagggtttgataaaatgtattacgactactttccggat

[0156] gccgccaaaatgattccgaagtgttcgacccagctgaaagcggtgaccgcgcattttcagacacacaccaccccgatcctgctttcaaataacttt

[0157] atagaaccgctggaaataaccaaagaaatttacgacctgaacaatccggaaaaagaaccgaagaaatttcagaccgcctacgcaaagaaaac

[0158] cggagatcaaaaaggttaccgcgaagccctgtgtaaatggattgattttacgcgcgattttctcagcaaatacaccaagaccacttcaatagatttg

[0159] agttcgctgcgtccgagctcccagtataaagacctgggcgaatattatgccgagctgaacccgttactgtaccacattagcttccagcgcattgcg

[0160] gaaaaagaaatcatggatgcagtcgaaactggaaagctttatctgttccagatttacaacaaggattttgcgaaaggccatcacggcaaaccgaa

[0161] tctacatactctctattggactggcctgttctctcccgaaaacttggccaagacctcaattaaactcaatggccaagccgagctgttctaccgtccga

[0162] aatcgcgcatgaagcggatggcccatcgtcttggggagaaaatgctcaataagaaactgaaggatcagaaaacaccgatccctgacacgctgt

[0163] atcaggaactttacgactatgtcaatcaccgtctgagtcacgatctgtcggacgaagcacgggccttactgccgaacgtaataaccaaagaggtg

[0164] tcacacgagatcattaaggatcgacgttttacctccgacaaattctttttccacgtaccgattacccttaattaccaggcggcgaactcaccctcgaa

[0165] atttaaccagcgcgtaaacgcgtacctgaaagagcacccggaaacccctattattgggattgaccgtggcgaacgcaatcttatctatataaccgt

[0166] aatcgactctaccggcaagattttagaacagcgtagcctaaacaccattcagcaatttgattaccagaaaaagttagataatcgggaaaaagaac

[0167] gtgtggcggcacggcaggcttggtcggtagttgggacgattaaggatcttaaacaaggttatttatcccaggttatacacgaaatcgtagacttaat

[0168] gatacattatcaagccgttgtggtgctggccaacctgaacttcggatttaagtcaaagcggacgggcattgccgaaaaggcggtgtatcagcagt

[0169] tcgagaagatgctgatcgataaactcaattgcttagtactaaaagattatcccgctgaaaaggtaggtggcgttctcaacccgtaccagcttacag

[0170] atcaatttaccagctttgcgaagatgggtacacagagcggttttctgttttatgttcccgcaccgtataccagcaaaatcgatcccctgaccggcttt

[0171] gttgatccttttgtatggaaaaccatcaaaaaccacgaaagccgcaaacatttcctggagggctttgattttcttcactacgatgtgaaaacaggcg

[0172] attttattctgcacttcaaaatgaatcgcaatcttagctttcagcgcggtctgcctgggtttatgccggcgtgggatattgtgttcgaaaagaatgaaa

[0173] cgcagttcgacgccaaaggcacaccgtttattgccgggaaacgtatagtaccggtgatcgaaaatcatcggtttacaggccgttatcgcgacctg

[0174] tacccggctaatgagctgattgcactgctcgaagaaaagggaattgtatttcgtgacggttccaatattctgcctaaactgttggaaaatgatgactc

[0175] gcacgcgattgataccatggtggctcttatccgcagcgttcttcaaatgcgtaactctaacgcggcgacgggggaagattatattaacagcccgg

[0176] tacgcgatctcaacggcgtctgttttgatagtcgcttccagaaccccgaatggccgatggacgcggacgccaacggagcataccatattgcgct

[0177] gaaaggccagctgttatgaatcatctcaaagaatccaaagacctcaaactacagaatggcatttcaaatcaggattggctcgcgtatattcaggaactgcgcaattga-3'

[0178] The crRNA is inserted into the vector plasmid pBbS8c-ddcpf1 (SEQ ID NO. 13) using Golden Gate Assembly. The coding sequence of the screening resistance gene CmR is preferably as shown in SEQ ID NO. 14 in the sequence listing.

[0179] SEQ ID NO.13:

[0180] 5'-ttgttcaggtcgtaaatttctttggttatttccagcggttctataaagttatttgaaagcaggatcggggtggtgtgtgtctgaaaatgcgcg gtcaccgctttcagctgggtcgaacacttcggaatcattttggcggcatccggaaagtagtcgtaatacattttatcaaacccttcactggtcttctccgtcggttcgaatgacagtgccttgtaacggcctttctgcttcggcataatgcccagataatacaggccgtttttcacaaataagatcgcaccgttgttcttctctttgttgacatcccaacctgacgctagggtcggcatttggaagttcaatttgaatttttccactgaataaggctttttggtcgcatagttgcgtg

[0181] ctttgttgtaaaagctcaacgacggttccatctccaattttataccggtcaggcgtgcgctaaattcagggtccacctcattagactcgtccaccgca

[0182] aaccagtccagcaagtggtacagtcccagtagagaatctaattgagatttcaggatttctttctcttcctgctttttcagcgtcgtcggcagaggctg

[0183] atctagggccgcgtgcgcatgtgagaggatttcgcttgtcttctgtttgaaagcctccgacagttccttaccggccgccgaaataatctcttgtaagt

[0184] taatatcttcatgtttgagggagcgctggactttctctttagctgacttggtgatctttccggtcagctcggaaattcgacgttcatacagggcatttct

[0185] cagcgtgtcccaatggtcgcacagcgcgctgctgatagtttccagctttttatgtgaaataaaaatatgggtgagatcaatgctgttcagttcgttaa

[0186] agagggcctcagccgtttccaggacgttttcattgcgcagtaacgttttatatttgcagaagctctgaattacttcttcatcgcttttaaattcctccagg

[0187] ataaagctcagtgtgttgcggtcggacagaatttgtttaaagagcggtataaatcgatgggggagagatgcaataatgtgcgctgtttcatcgttct

[0188] tttgaattgccaggttcagcacctcattcaggccttttatcttttccgttccagcttctcggctaataccacctaagagctgattatataggtcaatctgt

[0189] gtctgggtaagaagctgattatagaacgggaatgaaaacacttcttcaatgctggttgacacgaaaatgccaatcgccttcttaacattttcaaaatg

[0190] ttctcgcagtgatggcactgctgtgatcagacgagtaaaaatatgacaattttctttaaatttcggaaaattgtcttgaacaatacgatgcggaatgg

[0191] ccgtactaatatcttctgcgctaaacacgttcttgcgattctcgtagaacccggaaaaatacgtagtgaatttatcaaaagaacgcagcaaggcatt

[0192] ttcatgttccgtggtcgttacggtacctaactgctttaaatactttaccgttgaacagctccgctttaaacagacctttgtatatttcggcatgacgcttgtt

[0193] gatggcgtccgttagattatctgtcctaccaataaaatagtcatgaattgcattgcggtatgtagcctgctcctcgattaatgcattacgagtttcttct

[0194] gttttctccttacggtaagagtctatagccgcgcttaaattttcccaatctaactgcaccagttgcagacactggtctgcatacgtcttgtagatgcga

[0195] tcgataatcggtttaagttctttataatggtcgtttcgggccttatcctcttcaatgaagccttgctcttgaatgtgtttcagtgttttgccctggggaatc

[0196] agctcaaatctcagcgtcttgcttacctgataaagattggtaaaaccttcaaactgggtcatcacgacctcctacataaaaacagtttcatggattat

[0197] gaaccgcgcaagagaataacactgacatgcgaggcattttatgacggtgtagatcttttgaattcccaaaaaaacgggtatggagaaacagtag

[0198] agagttgcgataaaaagcgtcaggtagaatccgctaatcttatggataaaaatgctatggcatagcaaagtgtgacgccgtgcaaataatcaatgt

[0199] ggacttttctgccgtgattatagacacttttgttacgcgtttttgtcatggctttggtcccgctttgttacagaatgcttttaataagcggggttaccggtt

[0200] tggttagcgagaagagccagtaaaagacgcagtgacggcaatgtctgatgcaatatggacaattggtttcttctctgaatggcgggagtatgaaa

[0201] agtatggctgaagcgcaaaatgatcccctgctgccgggatactcgtttaatgcccatctggtggcgggtttaacgccgattgaggccaacggtta

[0202] tctcgatttttttatcgaccgaccgctgggaatgaaaggttatattctcaatctcaccattcgcggtcagggggtggtgaaaaatcagggacgaga

[0203] atttgtttgccgaccgggtgatattttgctgttcccgccaggagagattcatcactacggtcgtcatccggaggctcgcgaatggtatcaccagtgg

[0204] gtttactttcgtccgcgcgcctactggcatgaatggcttaactggccgtcaatatttgccaatacggggttctttcgcccggatgaagcgcaccagc

[0205] cgcatttcagcgacctgtttgggcaaatcattaacgccgggcaaggggaagggcgctattcggagctgctggcgataaatctgcttgagcaatt

[0206] gttactgcggcgcatggaagcgattaacgagtcgctccatccaccgatggataatcgggtacgcgaggcttgtcagtacatcagcgatcacctg

[0207] gcagacagcaattttgatatcgccagcgtcgcacagcatgtttgcttgtcgccgtcgcgtctgtcacatcttttccgccagcagttagggattagcg

[0208] tcttaagctggcgcgaggaccaacgtatcagccaggcgaagctgcttttgagcaccacccggatgcctatcgccaccgtcggtcgcaatgttgg

[0209] ttttgacgatcaactctatttctcgcgggtatttaaaaaatgcaccggggccagcccgagcgagttccgtgccggttgtgaagaaaaagtgaatga

[0210] tgtagccgtcaagttgtcataagacgtcgatatctggcgaaaatgagacgttgatcggcacgtaagaggttccaactttcaccataatgaaataag

[0211] atcactaccgggcgtattttttgagttatcgagattttcaggagctaaggaagctaaaatggagaaaaaaatcactggatataccaccgttgatatat

[0212] cccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaag

[0213] accgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaatttcgtatggcaatgaaaga

[0214] cggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgac

[0215] gatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcg

[0216] tctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattata

[0217] cgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatcatgccgtttgtgatggcttccatgtcggcagaatgcttaatgaattacaac

[0218] agtactgcgatgagtggcagggcggggcgtaatttgatatcgagctcgcttggactcctgttgatagatccagtaatgacctcagaactccatctg

[0219] gatttgttcagaacgctcggttgccgccgggcgttttttattggtgagaatccaagcactagggacagtaagacgggtaagcctgttgatgatacc

[0220] gctgccttactgggtgcattagccagtctgaatgacctgtcacgggataatccgaagtggtcagactggaaaatcagagggcaggaactgctga

[0221] acagcaaaaagtcagatagcaccacatagcagacccgccataaaacgccctgagaagcccgtgacgggcttttcttgtattatgggtagtttcctt

[0222] gcatgaatccataaaaggcgcctgtagtgccatttacccccattcactgccagagccgtgagcgcagcgaactgaatgtcacgaaaaagacag

[0223] cgactcaggtgcctgatggtcggagacaaaaggaatattcagcgatttgcccgagcttgcgagggtgctacttaagcctttagggttttaaggtct

[0224] gttttgtagaggagcaaacagcgtttgcgacatccttttgtaatactgcggaactgactaaagtagtgagttatacacagggctgggatctattctttt

[0225] tatctttttttattctttctttattctataaattataaccacttgaatataaacaaaaaaaacacacaaaggtctagcggaatttacagagggtctagcaga

[0226] atttacaagttttccagcaaaggtctagcagaatttacagatacccagatcacccgggaaaaggactagtaattatcattgactagcccatctcaatt

[0227] ggtatagtgattaaaatcacctagaccaattgagatgtatgtctgaattagttgttttcaaagcaaatgaactagcgattagtcgctatgacttaacgg

[0228] agcatgaaaccaagctaattttatgctgtgtggcactactcaaccccacgattgaaaaccctacaaggaaagaacggacggtatcgttcacttata

[0229] accaatacgctcagatgatgaacatcagtagggaaaatgcttatggtgtattagctaaagcaaccagagagctgatgacgagaactgtggaaat

[0230] caggaatccttggttaaaggctttgagattttccagtggacaaactatgccaagttctcaagcgaaaaattagaattagtttttagtgaagagatatt

[0231] gccttatcttttccagttaaaaaaattcataaaatataatctggaacatgttaagtcttttgaaaacaaatactctatgaggatttatgagtggttattaaa

[0232] agaactaacacaaaagaaaactcacaaggcaaatatagagattagccttgatgaatttaagttcatgttaatgcttgaaaataactaccatgagttta

[0233] aaaggcttaaccaatgggttttgaaaccaataagtaaagatttaaacacttacagcaatatgaaattggtggttgataagcgaggccgcccgactg

[0234] atacgttgattttccaagttgaactagatagacaaatggatctcgtaaccgaacttgagaacaaccagataaaaatgaatggtgacaaaataccaa

[0235] caaccattacatcagattcctacctacataacggactaagaaaaacactacacgatgctttaactgcaaaaattcagctcaccagttttgaggcaaa

[0236] atttttgagtgacatgcaaagtaagtatgatctcaatggttcgttctcatggctcacgcaaaaacaacgaaccacactagagaacatactggctaaa

[0237] tacggaaggatctgaggttcttatggctcttgtatctatcagtgaagcatcaagactaacaaacaaaagtagaacaactgttcaccgttacatatca

[0238] aagggaaaactgtccatatgcacagatgaaaacggtgtaaaaaagatagatacatcagagcttttacgagtttttggtgcattcaaagctgttcacc

[0239] atgaacagatcgacaatgtaacagatgaacagcatgtaacacctaatagaacaggtgaaaccagtaaaacaaagcaactagaacatgaaattg

[0240] aacacctgagacaacttgttacagctcaacagtcacacatagacagcctgaaacaggcgatgctgcttatcgaatcaaagctgccgacaacacg

[0241] ggagccagtgacgcctcccgtggggaaaaaatcatggcaattctggaagaaatagcgctttcagccggcaaaccggctgaagccggatctgc

[0242] gattctgataacaaactagcaacaccagaacagcccgtttgcgggcagcaaaacccgtaccctaggtataaacgcagaaaggcccacccgaa

[0243] ggtgagccagtgtgactctagtagagagcgttcaccgacaaacaacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttga

[0244] tgcctggagatccttactcgagtttggatccgagtttttgttcgggcccaagcttcaaaaaaaatctacaagagtagaaattgaaattgttatccgctc

[0245] acaattccacacattatacgagccggatgattaattgtcaacagctcatttaggtgtagacatcaagttaccaaagttgcgcagcgaaaaaacccc

[0246] gccgaagcggggttttttgcggtccatgagggtgaaggtcacatggactatcaattgcgcagttcctgaatatacgcgagccaatcctgatttgaa

[0247] atgccattctgtagtttgaggtctttggattctttgagatgattcagtaacagctggcctttcagcgcaatatggtatgctccgttggcgtccgcgtcc

[0248] atcggccattcggggttctggaagcgactatcaaaacagacgccgttgagatcgcgtaccgggctgttaatataatcttcccccgtcgccgcgtt

[0249] agagttacgcatttgaagaacgctgcggataagagccaccatggtatcaatcgcgtgcgagtcatcattttccaacagtttaggcagaatattgga

[0250] accgtcacgaaatacaattcccttttcttcgagcagtgcaatcagctcattagccgggtacaggtcgcgataacggcctgtaaaccgatgattttcg

[0251] atcaccggtactatacgtttcccggcaataaacggtgtgcctttggcgtcgaactgcgtttcattcttttcgaacacaatatcccacgccggcataaa

[0252] cccaggcagaccgcgctgaaagctaagattgcgattcattttgaagtgcagaataaaatcgcctgttttcacatcgtagtgaagaaaatcaaagcc

[0253] ctccaggaaatgtttgcggctttcgtggtttttgatggttttccatacaaaaggatcaacaaagccggtcaggggatcgattttgctggtatacggtg

[0254] cgggaacataaaacagaaaaccgctctgtgtacccatcttcgcaaagctggtaaattgatctgtaagctggtacgggttgagaacgccacctacc

[0255] ttttcagcgggataatcttttagtactaagcaattgagtttatcgatcagcatcttctcgaactgctgatacaccgccttttcggcaatgcccgtccgct

[0256] ttgacttaaatccgaagttcaggttggccagcaccacaacggcttgataatgtatcattaagtctacgatttcgtgtataacctgggataaataacctt

[0257] gtttaagatccttaatcgtcccaactaccgaccaagcctgccgtgccgccacacgttctttttcccgattatctaactttttctggtaatcaaattgctg

[0258] aatggtgtttaggctacgctgttctaaaatcttgccggtagagtcgattacggttatatagataagattgcgttcgccacggtcaatcccaataatag

[0259] gggtttccgggtgctctttcaggtacgcgtttacgcgctggttaaatttcgagggtgagttcgccgcctggtaattaagggtaatcggtacgtggaa

[0260] aaagaatttgtcggaggtaaaacgtcgatccttaatgatctcgtgtgacacctctttggttattacgttcggcagtaaggcccgtgcttcgtccgaca

[0261] gatcgtgactcagacggtgattgacatagtcgtaaagttcctgatacagcgtgtcagggatcggtgttttctgatccttcagtttcttattgagcatttt

[0262] ctccccaagacgatgggccatccgcttcatgcgcgatttcggacggtagaacagctcggcttggccattgagtttaattgaggtcttggccaagtt

[0263] ttcgggagagaacaggccagtccaatagagagtatgtagattcggtttgccgtgatggcctttcgcaaaatccttgttgtaaatctggaacagata

[0264] aagctttccagtttcgactgcatccatgatttctttttccgcaatgcgctggaagctaatgtggtacagtaacgggttcagctcggcataatattcgcc

[0265] caggtctttatactgggagctcggacgcagcgaactcaaatctattgaagtggtcttggtgtatttgctgagaaaatcgcgcgtaaaatcaatccatttacacagggcttcgcggtaacctttttgatctccggttttctttgcgtaggcggtctgaaatttcttcggttctttttccgga-3'

[0266] SEQ ID NO.14:

[0267] 5'-ttacgccccgccctgccactcatcgcagtactgttgtaattcattaagcattctgccgacatggaagccatcacaaacggcatgatgaa cctgaatcgccagcggcatcagcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattggctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcactccagagcgatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctttcattgccatacgaaattccggatgagcattcatcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactgactgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatccagtgatttttttctccat-3'

[0268] The xseA4, sbcB3, sbcB6, and exoX4 gene sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the reverse complement sequence was also synthesized. The specific sequences are as follows:

[0269] xseA4 crRNA 5' end sequence (SEQ ID NO.15):

[0270] 5'-acgcaaccagcttccggtcactg-3'

[0271] sbcB3 crRNA 5' end sequence (SEQ ID NO.16):

[0272] 5'-acctaatgatgaatgacggtaag-3'

[0273] sbcB6 crRNA 5' end sequence (SEQ ID NO.17):

[0274] 5'-acgattacgaaacctttggcacg-3'

[0275] exoX4 crRNA 5' end sequence (SEQ ID NO.18):

[0276] 5'-tggcctgggatcaagtacagcaa-3'

[0277] Anneal the designed and synthesized crRNA primers and phosphorylate them according to the T4 PNK instructions. The annealing reaction system consists of: 10 μL Primer_F (10 μM), 10 μL Primer_R (10 μM), and 5 μL 5× Anneal Buffer. The annealing program is: 95°C for 10 min, 25°C for 10 s, ramp 0.1°C / s, and 12°C for 10 min.

[0278] The phosphorylation reaction system was as follows: 6 μL ddH2O, 2.5 μL 4 μM oligonucleotide duplex, 1 μL 10× T4 DNA ligase buffer (NEB), and 0.5 μL T4 PNK (Takara). The reaction was carried out at 37°C for 30 min.

[0279] After completion of the above reaction, Golden Gate Assembly was performed. The GGA reaction system consisted of: 1 μL 10× T4 DNA ligase buffer, 0.5 μL pBbS8c-ddcpf1 (empty), 1 μL annealed and phosphorylated product, 0.2 μL T4 DNA ligase, 0.2 μL BsaI-HF-v2, and 7.1 μL ddH2O. The reaction procedure was as follows: 24 cycles of 37°C for 2 minutes, 16°C for 3 minutes, followed by 37°C for 15 minutes, 85°C for 5 minutes, and 10°C forever.

[0280] Transform 5 μL of the resulting GGA product into E. coli TOP10 cells and plate them in TB medium supplemented with 50 μg / mL chloramphenicol. After the transformation solution has been absorbed by the solid culture plate, incubate the plate in an inverted position at 30°C overnight. Transformants grown on the culture plate were transferred and stored, and plasmids were extracted using the SanPrep Column-Based Plasmid DNA Miniprep Kit from Sangon Biotech (Shanghai) Co., Ltd. for subsequent experiments. Specific plasmid extraction procedures were followed according to the kit's instruction manual. The plasmids were then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing confirmation.

[0281] Figure 2 Schematic diagram of the CRISPRi inhibition system plasmid construction. The figure shows the insertion of four crRNAs, xseA4, sbcB3, sbcB6, and exoX4, into the empty plasmid pBbS8c-ddcpf1 via Golden Gate assembly.

[0282] (1.2) RNA extraction and RT-qPCR verification of key gene inhibition effects

[0283] The pBbS8c-ddcpf1-Δ (SEQ ID NO. 1) plasmid sequenced in 1.1 was transformed into E. coli MG1655 and cultured overnight in TB medium supplemented with Cm on a shaker at 30°C. The next day, the bacteria were cultured in a 250 mL Erlenmeyer flask containing 100 mL of TB medium supplemented with Cm at a 1:50 ratio. After 1 hour, arabinose and IPTG were added. When the OD600 reached 0.5-0.6, the bacteria were chilled on ice for 10 minutes. Subsequently, 3 mL of the bacteria were centrifuged, the supernatant discarded, and the bacteria were resuspended in 1 mL of Buffer RL and transferred to Lysing Matrix B. The bacteria were then lysed using FastPrep and centrifuged at 12,000 g for 10 minutes in a refrigerated centrifuge. RNA was then purified according to the instructions of the TaKaRa MiniBEST Universal RNA Extraction Kit. Finally, the quality of the extracted RNA was judged by agarose gel electrophoresis. Clear 23S rRNA, 16S rRNA, and 5S rRNA should be observed before it could be used in subsequent experiments.

[0284] Use the total RNA purified in the previous step, Reverse transcription was performed according to the instructions for One-Step gDNA Removal and cDNA Synthesis SuperMix. Primers for qPCR were synthesized by Sangon Biotechnology Co., Ltd., and the specific sequences were designed as follows:

[0285] qPCR_sbcB_F(SEQ ID NO.19):5'-gcgctgggatttactggatg-3'

[0286] qPCR_sbcB_R(SEQ ID NO.20):5'-cagctttgccatcgcaatag-3'

[0287] qPCR_xseA_F(SEQ ID NO.21):5'-agtgagccgtaatcagcaag-3'

[0288] qPCR_xseA_R(SEQ ID NO.22):5'-ttccagcgcaaagctcattc-3'

[0289] qPCR_xseB_F(SEQ ID NO.23):5'-atgccgaagaaaaatgaggc-3'

[0290] qPCR_xseB_R(SEQ ID NO.24):5'-tcagacagcagaatttgtacgc-3'

[0291] qPCR_hcaT_F(SEQ ID NO.25):5'-ccgtttacaggcggtttat-3'

[0292] qPCR_hcaT_R(SEQ ID NO.26):5'-ccgtggcccagatattgatac-3'

[0293] The qPCR reaction system was as follows: ChamQ Universal SYBR qPCR Master Mix (10 μL), H2O (7 μL), Primer 1 (10 μM) (1 μL), Primer 2 (10 μM) (1 μL), and cDNA (1 μL). The following program was run in a qPCR instrument: 95°C for 30 s; 95°C for 10 s, 60°C for 30 s, 40 cycles; 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. LightCycler 96 software was then used to analyze melting curves and Ct values. The transcript levels of the target genes were then analyzed using the Ct difference comparison method (Schmittgen and Livak, 2008), using the hcaT gene as an internal reference.

[0294] Figure 3 The figure shows the transcriptional levels of genes involved in suppressing prime editing by CRISPRi. As shown in the figure, CRISPRi significantly suppressed the transcriptional levels of these genes.

[0295] Example 2 - Construction of the expression plasmid PEE1[blaZ-p15a-araC] for PE protein in prime editing

[0296] The sequence of PEE1[blaZ-p15a-araC] is SEQ ID NO. 2, and its specific construction method is as follows:

[0297] SEQ ID NO.2:

[0298]

[0299] tcacactttgctatgccatagcatttttatccataagattagcggatcctacctgacgctttttatcgcaactctctactgtttctccatacccgtttttttgg

[0300] gattcgagctctaaggaggttataaaaaatggataagaaatactcaataggcttagatatcggcacaaatagcgtcggatgggcggtgatcact

[0301] gatgaatataaggttccgtctaaaaagttcaaggttctgggaaatacagaccgccacagtatcaaaaaaaatcttataggggctcttttatttgacag

[0302] tggagagacagcggaagcgactcgtctcaaacggacagctcgtagaaggtatacacgtcggaagaatcgtatttgttatctacaggagattttttc

[0303] aaatgagatggcgaaagtagatgatagtttctttcatcgacttgaagagtcttttttggtggaagaagacaagaagcatgaacgtcatcctatttttg

[0304] gaaatatagtagatgaagttgcttatcatgagaaatatccaactatctatcatctgcgaaaaaaattggtagattctctgataaagcggatttgcgct

[0305] taatctatttggccttagcgcatatgattaagtttcgtggtcattttttgattgagggagatttaaatcctgataatagtgatgtggacaaactatttatcc

[0306] agttggtacaaacctacaatcaattatttgaagaaaaccctattaacgcaagtggagtagatgctaaagcgattctttctgcacgattgagtaaatca

[0307] agacgattagaaaatctcattgctcagctccccggtgagaagaaaaatggcttatttgggaatctcattgctttgtcattgggtttgacccctaatttta

[0308] aatcaaattttgatttggcagaagatgctaaattacagctttcaaaagatacttacgatgatgatttagataatttattggcgcaaattggagatcaata

[0309] tgctgatttgtttttggcagctaagaatttatcagatgctattttactttcagatatcctaagagtaaatactgaaataactaaggctcccctatcagcttc

[0310] aatgattaaacgctacgatgaacatcatcaagacttgactcttttaaaagctttagttcgacaacaacttccagaaaagtataaagaaatcttttttgat

[0311] caatcaaaaaacggatatgcaggttatattgatgggggagctagccaagaagaattttataaatttatcaaaccaattttagaaaaaatggatggta

[0312] ctgaggaattattggtgaaactaaatcgtgaagatttgctgcgcaagcaacggacctttgacaacggctctattccccatcaaattcacttgggtga

[0313] gctgcatgctattttgagaagacaagaagacttttatccatttttaaaagacaatcgtgagaagattgaaaaaatcttgacttttcgaattccttattatg

[0314] ttggtccattggcgcgtggcaatagtcgttttgcatggatgactcggaaatctgaagaaacaattaccccatggaattttgaagaagttgtcgataa

[0315] aggtgcttcagctcaatcatttattgaacgcatgacaaactttgataaaaaatcttccaaatgaaaaagtactaccaaaacatagtttgctttatgagtat

[0316] tttacggtttataacgaattgacaaaggtcaaatatgttactgaaggaatgcgaaaaccagcatttctttcaggtgaacagaagaaagccattgttg

[0317] atttactcttcaaaacaaatcgaaaagtaaccgttaagcaattaaaaagaagattattcaaaaaaaatagaatgttttgatagtgttgaaatttcaggagt

[0318] tgaagatagattttaatgcttcattaggttacctaccatgatttgctaaaaattattaaagataaagattttttggataatgaagaaaaatgaagatatcttag

[0319] aggattatgttttaacattgaccttatttgaagatagggagatgattgagaaagacttaaacatatgctcaccctttgatgataaggtgatgaaac

[0320] agcttaaacgtcgccgttatactggtggggacgtttgtctcgaaaattgattaatggtattagggataagcaatctggcaaaacaatattagatttttt

[0321] gaaatcagatggttttgccaatcgcaattttatgcagctgatccatgatgatagtttgacatttaaagaagacattcaaaaaagcacaagtgtctggac

[0322] aaggcgatagtttacatgaacatattgcaaatttaggctgtagccctgctattaaaaaaggtattttacagactgtaaaagttgttgatgaattggtca

[0323] aagtaatggggcggcataagccagaaaatatcgttattgaaatggcacgtgaaaatcagacaactcaaaagggccagaaaaattcgcgagagc

[0324] gtatgaaacgaatcgaagaaggtatcaaagaattaggaagtcagattcttaaagagcatcctgttgaaaatactcaattgcaaaatgaaaagctct

[0325] atctctattatctccaaaatggagaagacatgtatgtggaccaagaattatagattaatcgtttaagtgattatgatgtcgatGCTattgttccacaa

[0326] agtttccttaaagacgattcaatagacaataaggtcttaacgcgttctgataaaaaatcgtggtaaatcggataacgttccaagtgaagaagtagtca

[0327] aaaagatgaaaaactattggagacaacttctaaacgccaagttaatcactcaacgtaagtttgataatttaacgaaaagctgaacgtggaggtttga

[0328] gtgaacttgataaagctgggttttatcaaacgccaattggttgaaactcgccaaatcactaagcatgtggcacaaattttggatagtcgcatgaatact

[0329] aaatacgatgaaaatgataaacttattcgagaggttaaagtgattaccttaaaatctaaattagtttctgacttccgaaaagatttccaattctataaaagt

[0330] acgtgagattaacaattaccatcatgcccatgatgcgtatctaaatgccgtcgttggaactgctttgattaagaaatatccaaaacttgaatcggagt

[0331] ttgtctatggtgattataaagtttatgatgttcgtaaaatgattgctaagtctgagcaagaaataggcaaagcaaccgcaaaatatttcttttactctaat

[0332] atcatgaacttcttcaaaacagaaattacacttgcaaatggagagattcgcaaacgccctctaatcgaaactaatggggaaactggagaaattgtc

[0333] tgggataaagggcgagattttgccacagtgcgcaaagtattgtccatgccccaagtcaatattgtcaagaaaacagaagtacagacaggcggat

[0334] tctccaaggagtcaattttaccaaaaagaaattcggacaagcttattgctcgtaaaaaagactgggatccaaaaaaatatggtggttttgatagtcc

[0335] aacggtagcttattcagtcctagtggttgctaaggtggaaaaagggaaatcgaagaagttaaaatccgttaaagagttactagggatcacaattat

[0336] ggaaagaagttcctttgaaaaaaatccgattgactttttagaagctaaaggatataaggaagttaaaaaagacttaatcattaaactacctaaatata

[0337] gtctttttgagttagaaaacggtcgtaaacggatgctggctagtgccggagaattacaaaaaggaaatgagctggctctgccaagcaaatatgtg

[0338] aattttttatatttagctagtcattatgaaaagttgaagggtagtccagaagataacgaacaaaaacaattgtttgtggagcagcataagcattattta

[0339] gatgagattattgagcaaatcagtgaattttctaagcgtgttattttagcagatgccaatttagataaagttcttagtgcatataacaaacatagagaca

[0340] aaccaatacgtgaacaagcagaaaatattattcatttatttacgttgacgaatcttggagctcccgctgcttttaaatattttgatacaacaattgatcgt

[0341] aaacgatatacgtctacaaaagaagttttagatgccactcttatccatcaatccatcactggtctttatgaaacacgcattgatttgagtcagctagga

[0342] ggtgacAGCGGCGGTTCTTCCGGCGGCTCCAGCGGTTCCGAAACTCCGGGTACCTCTGAAT

[0343] CTGCGACCCCGGAATCTTCTGGCGGCAGCTCTGGCGGTAGCAGCACCCTGAACATCGA

[0344] AGATGAATATCGTCTGCACGAAACCTCTAAAGAGCCGGACGTTTCCCTGGGCAGCACC

[0345] TGGCTGTCCGATTTCCCGCAGGCGTGGGCGGAAACCGGCGGCATGGGTCTGGCGGTTC

[0346] GCCAGGCGCCGCTGATCATCCCGCTGAAAGCGACCTCTACCCCGGTAAGCATCAAACA

[0347] GTATCCGATGAGCCAGGAAGCGCGTTTAGGTATCAAACCGCATATTCAGCGCCTGCTG

[0348] GATCAGGGTATTCTGGTGCCGTGCCAGTCTCCGTGGAACACTCCACTGCTTCCGGTCAA

[0349] AAAACCGGGTACCAACGACTACCGTCCGGTACAGGATCTGCGTGAAGTTAACAAACGT

[0350] GTTGAAGATATCCATCCGACCGTACCGAACCCGTATAACCTGCTGTCTGGCCTGCCGCC

[0351] GAGCCACCAGTGGTACACCGTGCTGGATTTGAAAGATGCGTTTTTCTGTCTGCGCCTGC

[0352] ATCCGACCTCCCAACCGCTGTTCGCCTTCGAATGGCGTGATCCGGAGATGGGTATTAGC

[0353] GGCCAGCTGACTTGGACTCGTCTGCCGCAGGGCTTCAAAAACAGCCCGACGCTCTTCA

[0354] ACGAAGCGCTGCATCGTGACCTGGCCGATTTCCGTATCCAGCACCCGGATCTGATCTTG

[0355] CTGCAGTACGTGGACGACCTGCTGCTGGCGGCAACGTCGGAACTGGACTGTCAGCAGG

[0356] GCACTCGTGCACTGCTGCAGACCCTGGGTAACCTGGGTTATCGTGCATCTGCTAAAAA

[0357] AGCCCAGATCTGTCAGAAACAGGTTAAATACCTCGGCTACTTGCTGAAAGAAGGCCAG

[0358] CGCTGGCTTACCGAAGCTCGTAAAGAGACTGTGATGGGTCAGCCGACGCCGAAAACCC

[0359] CGCGCCAACTGCGTGAATTCCTGGGTAAAGCTGGCTTCTGCCGTCTGTTCATTCCGGGC

[0360] TTTGCTGAAATGGCGGCTCCGCTGTATCCACTGACCAAACCGGGTACCCTGTTCAACTG

[0361] GGGCCCTGACCAGCAGAAAGCATACCAGGAAATTAAACAGGCTCTTCTGACCGCGCCG

[0362] GCACTGGGCCTGCCGGATCTGACCAAACCGTTCGAACTGTTTGTTGATGAAAAACAGG

[0363] GTTACGCGAAAGGCGTCCTGACCCAGAAACTGGGTCCGTGGCGCCGCCCGGTGGCGTA

[0364] CCTGAGCAAAAAACTGGATCCGGTTGCAGCAGGTTGGCCGCCGTGCCTGCGCATGGTT

[0365] GCTGCGATTGCTGTGTTGACCAAAGATGCTGGTAAACTGACCATGGGTCAGCCGCTGG

[0366] TGATCCTGGCTCCGCACGCTGTTGAAGCGCTGGTAAAACAGCCGCCGGACCGCTGGTT

[0367] GTCTAACGCCCGTATGACCCACTATCAGGCTCTGCTGCTGGACACCGACCGTGTTCAGT

[0368] TCGGCCCAGTGGTGGCACTGAATCCAGCGACCCTGCTGCCACTGCCGGAAGAGGGTCT

[0369] GCAGCACAACTGCCTTGATATCCTGGCCGAAGCGCACGGTACCCGTCCGGACTTAACC

[0370] GATCAGCCGCTGCCGGATGCTGATCATACCTGGTATACCGACGGTTCCTCTTTGTTGCA

[0371] GGAAGGTCAGCGTAAAGCGGGTGCGGCCGTTACCACCGAAACTGAAGTAATTTGGGCC

[0372] AAGGCGCTCCCGGCGGGCACGTCCGCGCAGCGCGCTGAACTCATCGCACTGACCCAGG

[0373] CGTTGAAAATGGCAGAAGGCAAAAAACTGAATGTGTACACTGATTCCCGCTACGCGTT

[0374] CGCAACCGCTCACATCCACGGCGAAATCTACCGCCGTCGCGGCTGGCTGACTAGCGAA

[0375] GGTAAAGAAATTAAAAATAAAGATGAAATCCTGGCACTGCTGAAAGCACTGTTCCTGC

[0376] CGAAACGTCTGTCTATCATTCACTGCCCAGGCCACCAGAAGGGCCACTCCGCTGAAGC

[0377] ACGTGGTAACCGCATGGCTGATCAGGCAGCACGTAAAGCGGCAATTACCGAAACCCCG

[0378] GACACCTCTACCCTGCTGATCGAAAACTCTTCGCCGAGCGGTGGCTCTAAACGTACCGC

[0379] TGACGGCTCCGAATTTGAATAAcgcatcctcacgataatatccgggtaggcgcaatcactttcgtctactccgttacaaagc

[0380] gaggctgggtatttcccggcctttctgttatccgaaatccactgaaagcacagcggctggctgaggagataaataataaacgaggggctgtatgc

[0381] acaaagcatcttctgttgagttaagaacgagtatcgagatggcacatagccttgctcaaattggaatcaggtttgtgccaataccagtagaaacag

[0382] acgaagaatccatgggtatggTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACG

[0383] CTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCT

[0384] GGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTG

[0385] TTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCT

[0386] CCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGT

[0387] AACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCA

[0388] CTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTG

[0389] AAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTT

[0390] GGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGC

[0391] AAGAGATTACGCGCAGACCAAAACGATCTCAAgtcgcaaacgctgtttgctcctctacaaaacagaccttaaaac

[0392] cctaaaggcttaagtagcaccctcgcaagctcggttgcggccgcaatcgggcaaatcgctgaatattccttttgtctccgaccatcaggcacctg

[0393] agtcgctgtctttttcgtgacattcagttcgctgcgctcacggctctggcagtgaatgggggtaaatggcactacaggcgccttttatggattcatgc

[0394] aaagcaactacccataatacaagaaaagcccgtcacgggcttctcagggcgttttatggcgggtctgctatgtggtgctatctgacttttgctgttc

[0395] agcagttcctgccctctgattttccagtctgaccacttcggattatcccgtgacaggtcattcagactggctaatgcacccagtaaggcagcggtat

[0396] catcaacggggtctgacgctcagtggaacgaaaactcacgttaaagggattttggtcatgagattatcaaaaaaggatcttcacctagatccttttaaa

[0397] ttaaaaaatgaagttttaaatcaatctaaagttatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgt

[0398] ctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacggagggcttaccatctggccccaggtgctgcaatgataccgc

[0399] gagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcct

[0400] ccatccagtctattaattgttgccgggaagctagagtaaggttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtg

[0401] tcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagct

[0402] ccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccg

[0403] taagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacggga

[0404] taataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatc

[0405] cagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgcc

[0406] gcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcg-3'

[0407] The PE protein expression gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd., the coding sequence of the PE protein is shown in SEQ ID NO.4, the amino acid sequence of the PE protein is shown in SEQ ID NO.3, the p15a replicon sequence was amplified from the pET28a (SEQ ID NO.27) plasmid, and the CbR resistance gene was amplified from the pKD46_Cas9 (SEQ ID NO.28) plasmid, and its sequence is SEQ ID NO.29. The p15a replicon sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and its sequence is SEQ ID NO.30. The SpCas9_M-MLVmut gene was amplified using the SpCas9_M-MLVmut gene sequence as a template, and the SpCas9_M-MLVmut gene sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and its sequence is SEQ ID NO.31.

[0408] SEQ ID NO.3:

[0409]

[0410] SEQ ID NO.4:

[0411]

[0412] aaaggtcaaatatgttactgaaggaatgcgaaaaccagcatttctttcaggtgaacagaagaaagccattgttgatttactcttcaaaacaaatcga

[0413] aaagtaaccgttaagcaattaaaagaagattatttcaaaaaaatagaatgttttgatagtgttgaaatttcaggagttgaagatagatttaatgcttcat

[0414] taggtacctaccatgatttgctaaaaattattaaagataaagattttttggataatgaagaaaatgaagatatcttagaggatattgttttaacattgacc

[0415] ttattgaagatagggagatgattgaggaaagacttaaaacatatgctcacctctttgatgataaggtgatgaaacagcttaaacgtcgccgttatac

[0416] tggttggggacgtttgtctcgaaaattgattaatggtattagggataagcaatctggcaaaacaatattagattttttgaaatcagatggttttgccaat

[0417] cgcaattttatgcagctgatccatgatgatagtttgacatttaaagaagacattcaaaaagcacaagtgtctggacaaggcgatagtttacatgaac

[0418] atattgcaaatttagctggtagccctgctattaaaaaaggtattttacagactgtaaaagttgttgatgaattggtcaaagtaatggggcggcataag

[0419] ccagaaaatatcgttattgaaatggcacgtgaaaatcagacaactcaaaagggccagaaaaattcgcgagagcgtatgaaacgaatcgaagaa

[0420] ggtatcaaagaattaggaagtcagattcttaaagagcatcctgttgaaaatactcaattgcaaaatgaaaagctctatctctattatctccaaaatgg

[0421] aagagacatgtatgtggaccaagaattagatatatcgtttaagtgattatgatgtcgatGCTattgttccacaaagtttccttaaagacgattcaa

[0422] tagacaataaggtcttaacgcgttctgataaaaaatcgtggtaaatcggataacgttccaaggaagaaggtcaaaaaagatgaaaaaactattgga

[0423] gacaactttaaacgccaagttaatcactcaacgtaagtttgataatttaacgaaagctgaacgtggaggtttgagtgaacttgataaagctggtttt

[0424] atcaaacgccaattggttgaaactcgccaaatcactaagcatgtggcacaaattttggatagtcgcatgaatactaaatacgatgaaaatgataaa

[0425] cttattcgagaggttaaagtgattaccttaaaatctaaattagtttctgacttccgaaaagatttccaattctataaagtacgtgagattaacaattacca

[0426] tcatgcccatgatgcgtatctaaatgccgtcgttggaactgctttgattaagaaatatccaaaacttgaatcggagtttgtctatggtgattaaaagtt

[0427] tatgatgttcgtaaaatgattgctaagtctgagcaagaaataggcaaagcaaccgcaaaatttcttttactctaatatcatgaacttcttcaaaaca

[0428] gaaattacacttgcaaatggagagattcgcaaacgccctctaatcgaaactaatggggaaactggagaaattgtctgggataaagggcgagattt

[0429] tgccacagtgcgcaaagtattgtccatgccccaagtcaatattgtcaagaaaacagaagtacagacaggcggattctccaaggagtcaattttac

[0430] caaaaaagaaattcggacaagcttattgctcgtaaaaaagactgggatccaaaaaaatatggtggttttgatagtccaacggtagcttattcagtcct

[0431] agtggttgctaaggtggaaaaagggaaatcgaagaagttaaaatccgttaaagagttactagggatcacaattatggaaagaagttcctttgaaa

[0432] aaaatccgattgactttttagaagctaaaggatataaggaagttaaaaaagacttaatcattaaactacctaaatatagtctttttgagttagaaaacg

[0433] gtcgtaaacggatgctggctagtgccggagaattacaaaaaggaaatgagctggctctgccaagcaaatatgtgaattttttatatttagctagtca

[0434] ttatgaaaagttgaagggtagtccagaagataacgaacaaaaacaattgtttgtggagcagcataagcattattgatgagattattgagcaaatc

[0435] agtgaattttctaagcgtgttattttagcagatgccaatttagataaagttcttagtgcatataacaaacatagagacaaaccaatacgtgaacaagc

[0436] agaaaatattattcatttatttacgttgacgaatcttggagctcccgctgcttttaaatattttgatacaacaattgatcgtaaacgatatacgtctacaaa

[0437] agaagttttagatgccactcttatccatcaatccatcactggtctttatgaaacacgcattgatttgagtcagctaggaggtgacAGCGGCGG

[0438] TTCTTCCGGCGGCTCCAGCGGTTCCGAAACTCCGGGTACCTCTGAATCTGCGACCCCGG

[0439] AATCTTCTGGCGGCAGCTCTGGCGGTAGCAGCACCCTGAACATCGAAGATGAATATCG

[0440] TCTGCACGAAACCTCTAAAGAGCCGGACGTTTCCCTGGGCAGCACCTGGCTGTCCGATT

[0441] TCCCGCAGGCGTGGGCGGAAACCGGCGGCATGGGTCTGGCGGTTCGCCAGGCGCCGCT

[0442] GATCATCCCGCTGAAAGCGACCTCTACCCCGGTAAGCATCAAACAGTATCCGATGAGC

[0443] CAGGAAGCGCGTTTAGGTATCAAACCGCATATTCAGCGCCTGCTGGATCAGGGTATTC

[0444] TGGTGCCGTGCCAGTCTCCGTGGAACACTCCACTGCTTCCGGTCAAAAAACCGGGTAC

[0445] CAACGACTACCGTCCGGTACAGGATCTGCGTGAAGTTAACAAACGTGTTGAAGATATC

[0446] CATCCGACCGTACCGAACCCGTATAACCTGCTGTCTGGCCTGCCGCCGAGCCACCAGT

[0447] GGTACACCGTGCTGGATTTGAAAGATGCGTTTTTCTGTCTGCGCCTGCATCCGACCTCC

[0448] CAACCGCTGTTCGCCTTCGAATGGCGTGATCCGGAGATGGGTATTAGCGGCCAGCTGA

[0449] CTTGGACTCGTCTGCCGCAGGGCTTCAAAAACAGCCCGACGCTCTTCAACGAAGCGCT

[0450] GCATCGTGACCTGGCCGATTTCCGTATCCAGCACCCGGATCTGATCTTGCTGCAGTACG

[0451] TGGACGACCTGCTGCTGGCGGCAACGTCGGAACTGGACTGTCAGCAGGGCACTCGTGC

[0452] ACTGCTGCAGACCCTGGGTAACCTGGGTTATCGTGCATCTGCTAAAAAAGCCCAGATC

[0453] TGTCAGAAACAGGTTAAATACCTCGGCTACTTGCTGAAAGAAGGCCAGCGCTGGCTTA

[0454] CCGAAGCTCGTAAAGAGACTGTGATGGGTCAGCCGACGCCGAAAACCCCGCGCCAACT

[0455] GCGTGAATTCCTGGGTAAAGCTGGCTTCTGCCGTCTGTTCATTCCGGGCTTTGCTGAAA

[0456] TGGCGGCTCCGCTGTATCCACTGACCAAACCGGGTACCCTGTTCAACTGGGGCCCTGAC

[0457] CAGCAGAAAGCATACCAGGAAATTAAACAGGCTCTTCTGACCGCGCCGGCACTGGGCC

[0458] TGCCGGATCTGACCAAACCGTTCGAACTGTTTGTTGATGAAAAACAGGGTTACGCGAA

[0459] AGGCGTCCTGACCCAGAAACTGGGTCCGTGGCGCCGCCCGGTGGCGTACCTGAGCAAA

[0460] AAACTGGATCCGGTTGCAGCAGGTTGGCCGCCGTGCCTGCGCATGGTTGCTGCGATTG

[0461] CTGTGTTGACCAAAGATGCTGGTAAACTGACCATGGGTCAGCCGCTGGTGATCCTGGC

[0462] TCCGCACGCTGTTGAAGCGCTGGTAAAACAGCCGCCGGACCGCTGGTTGTCTAACGCC

[0463] CGTATGACCCACTATCAGGCTCTGCTGCTGGACACCGACCGTGTTCAGTTCGGCCCAGT

[0464] GGTGGCACTGAATCCAGCGACCCTGCTGCCACTGCCGGAAGAGGGTCTGCAGCACAAC

[0465] TGCCTTGATATCCTGGCCGAAGCGCACGGTACCCGTCCGGACTTAACCGATCAGCCGCT

[0466] GCCGGATGCTGATCATACCTGGTATACCGACGGTTCCTCTTTGTTGCAGGAAGGTCAGC

[0467] GTAAAGCGGGTGCGGCCGTTACCACCGAAACTGAAGTAATTTGGGCCAAGGCGCTCCC

[0468] GGCGGGCACGTCCGCGCAGCGCGCTGAACTCATCGCACTGACCCAGGCGTTGAAAATG

[0469] GCAGAAGGCAAAAAACTGAATGTGTACACTGATTCCCGCTACGCGTTCGCAACCGCTC

[0470] ACATCCACGGCGAAATCTACCGCCGTCGCGGCTGGCTGACTAGCGAAGGTAAAGAAAT

[0471] TAAAAATAAAGATGAAATCCTGGCACTGCTGAAAGCACTGTTCCTGCCGAAACGTCTG

[0472] TCTATCATTCACTGCCCAGGCCACCAGAAGGGCCACTCCGCTGAAGCACGTGGTAACC

[0473] GCATGGCTGATCAGGCAGCACGTAAAGCGGCAATTACCGAAACCCCGGACACCTCTAC

[0474] CCTGCTGATCGAAAACTCTTCGCCGAGCGGTGGCTCTAAACGTACCGCTGACGGCTCCGAATTTGAATAA-3'

[0475] SEQ ID NO.27:

[0476] 5'-tggctggttggcgtactgttgtggtaaatacccatatggacagttttccctttgatatgtaacggtgaacagttgttctacttttgtttgttagtcttgatgcttcactgatagatacaagagCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACT

[0477] GACGGGGTGGTGCGTAACGGCAAAAGCACCGCCGGACATCAGCGCTAGCGGAGTGTA

[0478] TACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGA

[0479] GAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTC

[0480] CTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACG

[0481] AACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAG

[0482] GGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATC

[0483] TGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTC

[0484] CCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTC

[0485] CGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGT

[0486] TCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTAT

[0487] CCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGC

[0488] AGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTA

[0489] AACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAA

[0490] GAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTC

[0491] AGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAATCAG

[0492] ATAAAATATTTCTAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAGTCAG

[0493] CCCCATACGATATAAGTTGTGACGTaaaatgagacgttgatcggcacgtaagaggttccaactttcaccataatgaaata

[0494] agatcactaccgggcgtattttttgagttatcgagattttcaggagctaaggaagctaaaatggagaaaaaaatcactggatataccaccgttgata

[0495] tatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctgggtcatccaccggatca

[0496] attcccctgctcgcgcaggctgggtgccaagctctcgggtaacatcaaggcccgatccttggagcccttgccctcccgcacgatgatcgtgccg

[0497] tgatcgaaatccagatccttgacccgcagttgcaaaccctcactgatccggctcacggtaactgatgccgtatttgcagtaccagcgtacggccc

[0498] acagaatgatgtcacgctgaaaatgccggcctttgaatgggttcatgtgcagctccatcagcaaaaggggatgataagtttatcaccaccgactat

[0499] ttgcaacagtgccgttgatcgtgctatgatcgactgatgtcatcagcggtggagtgcaatgtcgtgcaatacgaatggcgaaaagccgagctcat

[0500] cggtcagcttctcaaccttggggttaccccccggcggtgtgctgctggtccacagctccttccgtagcgtccggcccctcgaagaatgggccacttg

[0501] gactgatcgaggccctgcgtgctgcgctgggtccgggagggacgctcgtcatgccctcgtggtcaggtctggacgacgagccgttcgatcctg

[0502] ccacgtcgcccgttacaccggaccttggagttgtctctgacacattctggcgcctgccaaatgtaaagcgcagcgcccatccatttgcctttgcgg

[0503] cagcggggccacaggcagagcagatcatctctgatccattgcccctgccacctcactcgcctgcaagcccggtcgccccgtgtccatgaactcg

[0504] atgggcaggtacttctcctcggcgtgggacacgatgccaacacgacgctgcatcttgccgagttgatggcaaaggttccctatggggtgccgag

[0505] acactgcaccattcttcaggatggcaagttggtacgcgtcgattatctcgagaatgaccactgctgtgagcgctttgccttggcggacaggtggct

[0506] caaggagaagagccttcagaaggaaggtccagtcggtcatgcctttgctcggttgatccgctcccgcgacattgtggcgacagccctgggtca

[0507] actgggccgagatccgttgatcttcctgcatccgccagaggcgggatgcgaagaatgcgatgccgctcgccagtcgattgggctgagctcatga

[0508] gcggagaacgagatgacgttggaggggcaaggtcgcgctgattgctggggcaacacgtgaaaggcgagatcaccaaggtagtcggcaaat

[0509] aatgtctaacggtacccggggatcctctagagtcgacctgcataatgtgcctgtcaaatggacgaagcagggattctgcaaaccctatgctactc

[0510] cgtcaagccgtcaattgtctgattcgttaccaattatgacaacttgacggctacatcattcactttttcttcacaaccggcacggaactcgctcgggct

[0511] ggccccggtgcattttttaaatacccgcgagaaatagagttgatcgtcaaaaccaacattgcgaccgacggtggcgataggcatccgggtggtg

[0512] ctcaaaagcagcttcgcctggctgatacgttggtcctcgcgccagcttaagacgctaatccctaactgctggcggaaaagatgtgacagacgcg

[0513] acggcgacaagcaaacatgctgtgcgacgctggcgatatcaaaattgctgtctgccaggtgatcgctgatgtactgacaagcctcgcgtacccg

[0514] attatccatcggtggatggagcgactcgttaatcgcttccatgcgccgcagtaacaattgctcaagcagatttatcgccagcagctccgaatagcg

[0515] cccttccccttgcccggcgttaatgatttgcccaaacaggtcgctgaaatgcggctggtgcgcttcatccgggcgaaagaaccccgtattggcaa

[0516] atattgacggccagttaagccattcatgccagtaggcgcgcggacgaaagtaaacccactggtgataccattcgcgagcctccggatgacgac

[0517] cgtagtgatgaatctctcctggcgggaacagcaaaatatcacccggtcggcaaacaaattctcgtccctgatttttcaccaccccctgaccgcga

[0518] atggtgagattgagaatataacctttcattcccagcggtcggtcgataaaaaaatcgagataaccgttggcctcaatcggcgttaaacccgccacc

[0519] agatgggcattaaacgagtatcccggcagcaggggatcattttgcgcttcagccatacttttcatactcccgccattcagagaagaaaccaattgt

[0520] ccatattgcatcagacattgccgtcactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgcttattaaaagcattctgtaacaaagc

[0521] gggaccaaagccatgacaaaaacgcgtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttgcacggcgtcacactttgcta

[0522] tgccatagcatttttatccataagattagcggatcctacctgacgctttttatcgcaactctctactgtttctccatacccgtttttttgggctagcgaaaa

[0523] gatgtttcgtgaagccgtcgacgcttataaaaaatggatattaatactgaaactgagatcaagcaaaagcattcactaaccccctttcctgttttccta

[0524] atcagcccggcatttcgcgggcgatattttcacagctatttcaggagttcagccatgaacgcttattacattcaggatcgtcttgaggctcagagct

[0525] gggcgcgtcactaccagcagctcgcccgtgaagagaaagaggcagaactggcagacgacatggaaaaaggcctgccccagcacctgtttga

[0526] atcgctatgcatcgatcatttgcaacgccacggggccagcaaaaaatccattacccgtgcgtttgatgacgatgttgagtttcaggagcgcatgg

[0527] cagaacacatccggtacatggttgaaaccattgctcaccaccaggttgatattgattcagaggtataaaacgaatgagtactgcactcgcaacgct

[0528] ggctgggaagctggctgaacgtgtcggcatggattctgtcgacccacaggaactgatcaccactcttcgccagacggcatttaaaggtgatgcc

[0529] agcgatgcgcagttcatcgcattactgatcgttgccaaccagtacggccttaatccgtggacgaaagaaatttacgcctttcctgataagcagaat

[0530] ggcatcgttccggtggtgggcgttgatggctggtcccgcatcatcaatgaaaaccagcagtttgatggcatggactttgagcaggacaatgaatc

[0531] ctgtacatgccggatttaccgcaaggaccgtaatcatccgatctgcgttaccgaatggatggatgaatgccgccgcgaaccattcaaaactcgcg

[0532] aaggcagagaaatcacggggccgtggcagtcgcatcccaaacggatgttacgtcataaagccatgattcagtgtgcccgtctggccttcggatt

[0533] tgctggtatctatgacaaggatgaagccgagcgcattgtcgaaaatactgcatacactgcagaacgtcagccggaacgcgacatcactccggtt

[0534] aacgatgaaaccatgcaggagattaacactctgctgatcgccctggataaaacatgggatgacgacttattgccgctctgttcccagatatttcgc

[0535] cgcgacattcgtgcatcgtcagaactgacacaggccgaagcagtaaaagctcttggattcctgaaacagaaagccgcagagcagaaggtggc

[0536] agcatgacaccggacattatcctgcagcgtaccgggatcgatgtgagagctgtcgaacagggggatgatgcgtggcacaaattacggctcgg

[0537] cgtcatcaccgcttcagaagttcacaacgtgatagcaaaaccccgctccggaaagaagtggcctgacatgaaaatgtcctacttccacaccctg

[0538] cttgctgaggtttgcaccggtgtggctccggaagttaacgctaaagcactggcctggggaaaacagtacgagaacgacgccagaaccctgttt

[0539] gaattcacttccggcgtgaatgttactgaatccccgatcatctatcgcgacgaaagtatgcgtaccgcctgctctcccgatggtttatgcagtgacg

[0540] gcaacggccttgaactgaaatgcccgtttacctcccgggatttcatgaagttccggctcggtggtttcgaggccataaagtcagcttacatggccc

[0541] aggtgcagtacagcatgtgggtgacgcgaaaaaatgcctggtactttgccaactatgacccgcgtatgaagcgtgaaggcctgcattatgtcgtg

[0542] attgagcgggatgaaaagtacatggcgagttttgacgagatcgtgccggagttcatcgaaaaaatggacgaggcactggctgaaattggttttgt

[0543] atttggggagcaatggcgatgacgcatcctcacgataataagcttcctgctgaacatcaaaggcaagaaaacatctgttgtcaaagacagcatcc

[0544] ttgaacaaggacaattaacagttaacaaataaaaacgcaaaagaaaatgccgatattgactaccggaagcagtgtgaccgtgtgcttctcaaatg

[0545] cctgattcaggctgtctatgtgtgactgttgagctgtaacaagttgtctcaggtgttcaatttcatgttctagttgctttgttttactggtttcacctgttcta

[0546] ttaggtgttacatgctgttcatctgttacattgtcgatctgttcatggtgaacagctttaaatgcaccaaaaactcgtaaaagctctgatgtatctatcttt

[0547] tttacaccgttttcatctgtgcatatagctatactgatttcgtcagactcacagtcaaacatgccggtcagttggcctggtgatggcgggatcgttgta

[0548] tatttcttgacaccttttcggcaccgccctaaaattctgcgtcctcataatatatgaggcgatttattacgtgtttacgaagcaaaagctaaaaccagg

[0549] agctatttaATGGTGCAGACGGAGATCCTGAAGGCGTTTCGGTTTGCGTTGGATCCGACGTCT

[0550] GTGCAGGTTGCTGCCCTTTCCCGGCATGCTGGTGCGGCCAGGTGGGCGTTCAACCATGC

[0551] TCTCGCGGCCAAGGTCGGCGCGCATGAGCGGTGGCGGGCGGAGGTAGCCAAGCTTGTC

[0552] GGGGACGGTGTTCCTGAGGAGCAGGCTCGTAGGCAGGTGCGCGTCCCGGTTCCGATGA

[0553] AGCCGGCGATCCAGAAGGCGTTGAACGCGGTCAAGGGTGACTCTCGCAAGGGCTTGGA

[0554] TGGTGCGTGCCCGTGGTGGCATGAGGTCAACACTTACGCGTTCCAGTCCGCTTTCATCG

[0555] ACGCGGACCAGGCGTGGAAGAACTGGTTGGACTCGTTGTCGGGGAAGCGGGTCGGCC

[0556] GCCGGGTGGGGTACCCGCGGTTCAAGAAGAAGGGGCGGGCCCGCGACTCGTTTCGCTT

[0557] GCACCACGACGTGAAGAAGCCAAGCATCCGGCTGGCCGGGTACCGGCGGTTGCGGCTG

[0558] CCGAGGATCGGGGAAGTCCGGCTTCATGACTCGGGGAAGCGTCTGGCACGGCTGATCG

[0559] ACCGAGGTGACGCGGTGGTGCAGTCGGTGACGGTGTCGCGGGGTGGCCATCGCTGGTA

[0560] TGCGTCTGTGCTGTGCAAGGTCACCGTGCAGGTACCGGATCGGCCGTCACGGCGCCAG

[0561] CGGGAACGAGGCGCTGTTGGGGTGGACCTTGGCGTTAAGGTCCTCGCGGCTTTGTCCA

[0562] AGCCACTGGTGGTCGACGACCCGTCGAGTGCCCTGGTGCGGAATCCGCAGCATCTGCG

[0563] GCAGGCCGAGCGGCGTCTACTGAAGGCTCAGCGGGCGTTGGCGCGTACACAAAAAGG

[0564] TTCAGCCCGACGCGAGAAGGCCAAGCGGCGAGTTGGTCGCGCGCATCACGAGGTTGCT

[0565] GTGCGCCGGCATGCTGCGCTCCATCAGATCACCAAGCGCCTGACCACCGGCTTCGCGG

[0566] TCGTCGCGCTTGAGGATCTGAACGTGGCCGGTATGACAAGGTCAGCGCGGGGAACAGT

[0567] GGCAGCGCCGGGAAAGAACGTGCGGCAGAAGGCAGGCCTGAACCGCGTCATCCTGGA

[0568] CTCCGCTCCAGCCGAGTTGCGTCGCCAGGTGAACTACAAGGCCACCTGGTATGGATCT

[0569] GAGCTGGCGGTCGCAGACCGCTGGTTCCCCTCGAGTAAGACCTGTTCGGGTTGCGGAT

[0570] GGCAAAACCCACACCTGAAGCTGTCCGACAGGGTGTTTCGTTGCACAGACTGCGGGCT

[0571] GGTGATGGACCGGGACATGAACGCGGCCCGGAACATCGAACGCCACGCAGTGCTGAT

[0572] CGATCGAAACGTCGCCTGCGATAGGCGGGAGACGCTAAACGCCCGTGGAGCATCCATA

[0573] AGACCAACCACCTCTCGGGGCGGTAGGCACGACGCATCGAAGCGGGAAGGCTCCGGC

[0574] GCTCGGCCTGAGTCACCTCAGCAGAGTGATCTGCTGACGCTCCCAACCTTGAATAACG

[0575] AAACGGCAACGCCTCCATAGCGGTGCAGGTCAATAAGGGTCGGCCCCACGCGTGTAGG

[0576] GAGCGATCGcgagaccattggtctcaGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGG

[0577] GCAACATGCTTCGGCATGGCGAATGGGACccaattattgAACACCCTAACGGGTGTTTTTTTGTTTctggtctacc-3'

[0578] SEQ ID NO.28:

[0579]

[0580] TGATGGCTGGTCCCGCATCATCAATGAAAACCAGCAGTTTGATGGCATGGACTTTGAG

[0581] CAGGACAATGAATCCTGTACATGCCGGATTTACCGCAAGGACCGTAATCATCCGATCT

[0582] GCGTTACCGAATGGATGGATGAATGCCGCCGCGAACCATTCAAAACTCGCGAAGGCAG

[0583] AGAAATCACGGGGCCGTGGCAGTCGCATCCCAAACGGATGTTACGTCATAAAGCCATG

[0584] ATTCAGTGTGCCCGTCTGGCCTTCGGATTTGCTGGTATCTATGACAAGGATGAAGCCGA

[0585] GCGCATTGTCGAAAATACTGCATACACTGCAGAACGTCAGCCGGAACGCGACATCACT

[0586] CCGGTTAACGATGAAACCATGCAGGAGATTAACACTCTGCTGATCGCCCTGGATAAAA

[0587] CATGGGATGACGACTTATTGCCGCTCTGTTCCCAGATATTTCGCCGCGACATTCGTGCA

[0588] TCGTCAGAACTGACACAGGCCGAAGCAGTAAAAGCTCTTGGATTCCTGAAACAGAAAG

[0589] CCGCAGAGCAGAAGGTGGCAGCATGACACCGGACATTATCCTGCAGCGTACCGGGATC

[0590] GATGTGAGAGCTGTCGAACAGGGGGATGATGCGTGGCACAAATTACGGCTCGGCGTCA

[0591] TCACCGCTTCAGAAGTTCACAACGTGATAGCAAAACCCCGCTCCGGAAAGAAGTGGCC

[0592] TGACATGAAAATGTCCTACTTCCACACCCTGCTTGCTGAGGTTTGCACCGGTGTGGCTC

[0593] CGGAAGTTAACGCTAAAGCACTGGCCTGGGGAAAACAGTACGAGAACGACGCCAGAA

[0594] CCCTGTTTGAATTCACTTCCGGCGTGAATGTTACTGAATCCCCGATCATCTATCGCGAC

[0595] GAAAGTATGCGTACCGCCTGCTCTCCCGATGGTTTATGCAGTGACGGCAACGGCCTTG

[0596] AACTGAAATGCCCGTTTACCTCCCGGGATTTCATGAAGTTCCGGCTCGGTGGTTTCGAG

[0597] GCCATAAAGTCAGCTTACATGGCCCAGGTGCAGTACAGCATGTGGGTGACGCGAAAAA

[0598] ATGCCTGGTACTTTGCCAACTATGACCCGCGTATGAAGCGTGAAGGCCTGCATTATGTC

[0599] GTGATTGAGCGGGATGAAAAGTACATGGCGAGTTTTGACGAGATCGTGCCGGAGTTCA

[0600] TCGAAAAAATGGACGAGGCACTGGCTGAAATTGGTTTTGTATTTGGGGAGCAATGGCG

[0601] ATGACGCATCCTCACGATAATATCCGGGTAGGCGCAATCACTTTCGTCTACTCCGTTAC

[0602] AAAGCGAGGCTGGGTATTTCCCGGCCTTTCTGTTATCCGAAATCCACTGAAAGCACAG

[0603] CGGCTGGCTGAGGAGATAAATAATAAACGAGGGGCTGTATGCACAAAGCATCTTCTGT

[0604] TGAGTTAAGAACGAGTATCGAGATGGCACATAGCCTTGCTCAAATTGGAATCAGGTTT

[0605] GTGCCAATACCAGTAGAAACAGACGAAGAATCCATGACGTCAGCTGACGAAATCATCC

[0606] TTGTGGAGCTTAGTAGGTTTAGCAAGATGGCAGCGCCTAAATGTAGAATGATAAAAGGA

[0607] TTAAGAGATTAATTTCCCTAAATTGATAAAACAAGCGTTTTGAAAGCGCTTGTTTTTT

[0608] TGGTTTGCAGTCAGAGTAGAATAGAAGTATCAAAAAGCACCGACTCGGTGCCACTT

[0609] TTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATGCTGTTTTGAATGGTTCCA

[0610] ACAAGATTATTTTATAACTTTTATAACAAATAATCAAGGAGAAATTCAAAGAAATTTAT

[0611] CAGCCATAAAACAATACTTAATACTATAGAATGATAACAAAATAAACTACTTTTTTAAA

[0612] AGAATTTTGTGTTATAATCTATTTATTATTAATTATTGGGTAATATTTTTTGAAGAGATA

[0613] TTTTGAAAGAAATTAAAGCATATTAAACTAATTTCGGAGGTCATTAAAACTATT

[0614] ATTGAAATCATCAAACTCATTATGGATTTAATTTAAACTTTTTATTTTTAGGAGGCAAAA

[0615] ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGG

[0616] TGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGA

[0617] CCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACA

[0618] GCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATC

[0619] GTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTC

[0620] TTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATC

[0621] CTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTAT

[0622] CATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTT

[0623] GGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATC

[0624] CTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTA

[0625] TTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCAC

[0626] GATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAA

[0627] AAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATC

[0628] AAATTTGATTTGGCAGAAGATGCTAAAATTACAGCTTTCAAAAGATACTTACGATGATG

[0629] ATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCT

[0630] AAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAAC

[0631] TAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGA

[0632] CTCTTTTAAAAGCTTTAGTTCGACAACACTTCCAGAAAAGTATAAAGAAATCTTTTTT

[0633] GATCAATCAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAA

[0634] TTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGT

[0635] GAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATT

[0636] CCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTA

[0637] TCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTT

[0638] ATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCT

[0639] GAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTC

[0640] AATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACT

[0641] ACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCA

[0642] AATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAAAGC

[0643] CATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAAATTAAAAGAA

[0644] GATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATA

[0645] GATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGAT

[0646] TTTTTGGATAATGAAGAAAAATGAAGATATCTTAGAGGGATATTGTTTTAACATTGACCTT

[0647] ATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGAT

[0648] GATAAGGTGAATGAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTC

[0649] GAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTT

[0650] GAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGA

[0651] CATTTAAAGAAGCATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGA

[0652] ACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAGGTATTTTACAGACTGTAA

[0653] AAGTTGTTGATGAATTGGTCAAAGTAATGGGCGGCATAAGCCAGAAATATCGTTAT

[0654] TGAAATGGCACGTGAAAATCAGACAACTCAAAAGGCCAGAAAAATTCGCGAGAGCG

[0655] TATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCAT

[0656] CCTGTTGAAAATACTCAAATTGCAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGG

[0657] AAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTC

[0658] GATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACATAAGGTCTTAAC

[0659] GCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAGTAGTCAAA

[0660] AAGATGAAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGT

[0661] TTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTT

[0662] ATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGG

[0663] ATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGT

[0664] GATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAG

[0665] TACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGA

[0666] ACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAA

[0667] AGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACC

[0668] GCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGC

[0669] AAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATT

[0670] GTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAG

[0671] TCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTT

[0672] ACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAA

[0673] ATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGG

[0674] AAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTAT

[0675] GGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAG

[0676] GAAGTTAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAA

[0677] ACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAGGAAATGAGCTGG

[0678] CTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAG

[0679] GGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATT

[0680] TAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCC

[0681] AATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAAC

[0682] AAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCCGCTGCTTTT

[0683] AAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAG

[0684] ATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGT

[0685] CAGCTAGGAGGTGACTGATGGCTGGTTGGCGTACTGTTGTGGTAAATACCCAGCTGTCT

[0686] AGAAGCTTAGATCTCCGCGGTGAAGGCATTACCCGGCGGGAATGCTTCAGCGGCGACC

[0687] GTGATGCGGTGCGTCGTCAGGCTACTGCGTATGCATTGCAGACCTTGTGGCAACAATTT

[0688] CTACAAAACACTTGATACTGTATGAGCATACAGTATAATTGCTTCAACAGAACATATTG

[0689] ACTATCCGGTATTCGTGGCCGTCCGGCCGCCCTGACTTCGCGAGGACTTCAATGGACGA

[0690] GAACAAGAAGCGCGCCCTGGCCGCGGCCCTGGGACAGATCGAACGCCAATTCGGCAA

[0691] AGGCGCGGTCATGCGCATGGGCGACCATGAGCGCCAGGCGATCCCGGCCATCTCCACC

[0692] GGCTCCCTGGGTCTGGACATCGCCCTCGGCATCGGCGGCCTGCCCAAGGGCCGGATCG

[0693] TCGAGATCTACGGTCCGGAATCCTCGGGCAAGACCACCCTGACCCTCTCGGTGATCGC

[0694] CGAGGCCCAGAAACAGGGCGCCACCTGTGCCTTCGTCGACGCCGAGCACGCGCTCGAT

[0695] CCCGACTATGCCGGCAAGCTGGGCGTCAACGTCGACGACCTGCTGGTCTCCCAGCCGG

[0696] ACACCGGCGAGCAGGCCCTGGAAATCACCGACATGCTGGTGCGCTCCAACGCGGTCGA

[0697] CGTGATCATCGTCGACTCCGTGGCCGCGCTGGTACCCAAGGCCGAGATCGAAGGCGAG

[0698] ATGGGCGACGCCCACGTCGGCCTGCAGGCACGCCTGATGTCCCAGGCGCTGCGCAAGA

[0699] TCACCGGCAATATCAAGAACGCCAACTGCCTGGTCATCTTCATCAACCAGATCCGCAT

[0700] GAAGATCGGCGTCATGTTCGGCAACCCGGAAACCACCACCGGCGGTAACGCACTGAAG

[0701] TTCTACGCCTCGGTCCGCCTGGACATCCGTCGTACCGGCGCGGTGAAGGAAGGCGACG

[0702] AGGTGGTGGGTAGCGAAACCCGCGTCAAGGTGGTGAAGAACAAGGTTTCCCCGCCGTT

[0703] CCGCCAGGCCGAGTTCCAGATCCTCTACGGTAAGGGCATCTACCGTACCGGCGAGATC

[0704] ATCGATCTGGGCGTGCAATTGGGCCTGGTCGAGAAGTCCGGCGCCTGGTACAGCTACC

[0705] AGGGCAGCAAGATCGGCCAGGGCAAGGCGAACGCCGCCAAGTACCTGGAAGACAATC

[0706] CGGAAATCGGTTCGGTACTGGAGAAGACCATTCGCGACCAGTTGCTGGCCAAGAGCGG

[0707] CCCGGTGAAGGCCGACGCCGAAGAAGTGGCTGACGCCGAAGCCGATTGAGGCCAATG

[0708] GCGATCGTGCTCGATACGCCCGTCGCCGTGCGGCGGGCGGCCATGGATCTGCTGGCGC

[0709] GCCGTGAGCACGGGCGCGCCGAGCTTTCCCGCAAGTTGCGCCAGCGTGCATGCTCTCG

[0710] AGGGCTAGCTTCATGGGTATGGACAGTTTTCCCTTTGATATGTAACGGTGAACAGTTGT

[0711] TCTACTTTTGTTTGTTAGTCTTGATGCTTCACTGATAGATACAAGAGCCATAAGAACCT

[0712] CAGATCCTTCCGTATTTAGCCAGTATGTTCTCTAGTGTGGTTCGTTGTTTTTGCGTGAGC

[0713] CATGAGAACGAACCATTGAGATCATACTTACTTTGCATGTCACTCAAAAATTTTGCCTC

[0714] AAAACTGGTGAGCTGAATTTTTGCAGTTAAAGCATCGTGTAGTGTTTTTCTTAGTCCGT

[0715] TAtGTAGGTAGGAATCTGATGTAATGGTTGTTGGTATTTTGTCACCATTCATTTTTATCT

[0716] GGTTGTTCTCAAGTTCGGTTACGAGATCCATTTGTCTATCTAGTTCAACTTGGAAAATC

[0717] AACGTATCAGTCGGGCGGCCTCGCTTATCAACCACCAATTTCATATTGCTGTAAGTGTT

[0718] TAAATCTTTACTTATTGGTTTCAAAACCCATTGGTTAAGCCTTTTAAACTCATGGTAGTT

[0719] ATTTTCAAGCATTAACATGAACTTAAATTCATCAAGGCTAATCTCTATATTTGCCTTGT

[0720] GAGTTTTCTTTTGTGTTAGTTCTTTTAATAACCACTCATAAATCCTCATAGAGTATTTGT

[0721] TTTCAAAAGACTTAACATGTTCCAGATTATATTTTATGAATTTTTTTAACTGGAAAAGA

[0722] TAAGGCAATATCTCTTCACTAAAAACTAATTCTAATTTTTCGCTTGAGAACTTGGCATA

[0723] GTTTGTCCACTGGAAAATCTCAAAGCCTTTAACCAAAGGATTCCTGATTTCCACAGTTC

[0724] TCGTCATCAGCTCTCTGGTTGCTTTAGCTAATACACCATAAGCATTTTCCCTACTGATGT

[0725] TCATCATCTGAaCGTATTGGTTATAAGTGAACGATACCGTCCGTTCTTTCCTTGTAGGGT

[0726] TTTCAATCGTGGGGTTGAGTAGTGCCACACAGCATAAAATTAGCTTGGTTTCATGCTCC

[0727] GTTAAGTCATAGCGACTAATCGCTAGTTCATTTGCTTTGAAAACAACTAATTCAGACAT

[0728] ACATCTCAATTGGTCTAGGTGATTTTAATCACTATACCAATTGAGATGGGCTAGTCAAT

[0729] GATAATTACTAGTCCTTTTCCTTTGAGTTGTGGGTATCTGTAAATTCTGCTAGACCTTTG

[0730] CTGGAAAACTTGTAAATTCTGCTAGACCCTCTGTAAATTCCGCTAGACCTTTGTGTGTT

[0731] TTTTTTGTTTATATTCAAGTGGTTATAATTTATAGAATAAAGAAAGAATAAAAAAAGAT

[0732] AAAAAGAATAGATCCCAGCCCTGTGTATAACTCACTACTTTAGTCAGTTCCGCAGTATT

[0733] ACAAAAGGATGTCGCAAACGCTGTTTGCTCCTCTACAAAACAGACCTTAAAACCCTAA

[0734] AGGCTTAAGTAGCACCCTCGCAAGCTCGGTTGCGGCCGCAATCGGGCAAATCGCTGAA

[0735] TATTCCTTTTGTCTCCGACCATCAGGCACCTGAGTCGCTGTCTTTTTCGTGACATTCAGT

[0736] TCGCTGCGCTCACGGCTCTGGCAGTGAATGGGGGTAAATGGCACTACAGGCGCCTTTT

[0737] ATGGATTCATGCAAGGAAACTACCCATAATACAAGAAAAGCCCGTCACGGGCTTCTCA

[0738] GGGCGTTTTATGGCGGGTCTGCTATGTGGTGCTATCTGACTTTTTGCTGTTCAGCAGTTC

[0739] CTGCCCTCTGATTTTCCAGTCTGACCACTTCGGATTATCCCGTGACAGGTCATTCAGAC

[0740] TGGCTAATGCACCCAGTAAGGCAGCGGTATCATCAACGGGGTCTGACGCTCAGTGGAA

[0741] CGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAG

[0742] ATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTG

[0743] GTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTC

[0744] GTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTA

[0745] CCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTT

[0746] ATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTA

[0747] TCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGT

[0748] TAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGT

[0749] TTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCC

[0750] ATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTT

[0751] GGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGC

[0752] CATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAG

[0753] TGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCAC

[0754] ATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTC

[0755] AAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGAT

[0756] CTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAAT

[0757] GCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTT

[0758] TTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACA-3'

[0759] SEQ ID NO.29:

[0760] 5'-ttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcat-3'

[0761] SEQ ID NO.30:

[0762] 5'-TTGAGATCGTTTTGGTCTGCGCGTAATCTCTTGCTCTGAAAACGAAAAAACCGCCTTGCAGGGCGGTTTTTCGAAGGTTCTCTGAGCTACCAACTCTTTGAACCGAGGTAACTGGCTTGGAGGAGCGCAGTCACCAAAACTTGTCCTTTCAGTTTAGCCTTAACCGGCGCATGACTTCAAGACTAACTCCTCTAAATCAATTACCAGTGGCTGCTGCCAGTGGTGCTTTTGCATGTCTTTCCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGACTGAACGGGGGGTTCGTGCATACAGTCCAGCTTGGAGCGAACTGCCTACCCGGAACTGAGTGTCAGGCGTGGAATGAGACAAACGCGGCCATAACAGCGGAATGACACCGGTAAACCGAAAGGCAGGAACAGGAGAGCGCACGAGGGAGCCGCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCACTGATTTGAGCGTCAGATTTCGTGATGCTTGTCAGGGGGGCGGAGCCTATGGAAA-3'

[0763] SEQ ID NO.31:

[0764] 5'-atggataagaaatactcaataggcttagatatcggcacaaatagcgtcggatgggcggtgatcactgatgaatataaggttccgtctaa

[0765] aaagttcaaggttctgggaaatacagaccgccacagtatcaaaaaaaatcttataggggctcttttatttgacagtggagagacagcggaagcga

[0766] ctcgtctcaaacggacagctcgtagaaggtatacacgtcggaagaatcgtatttgttatctacaggagattttttcaaatgagatggcgaaagtaga

[0767] tgatagtttctttcatcgacttgaagagtcttttttggtggaagaagacaagaagcatgaacgtcatcctatttttggaaatatagtagatgaagttgct

[0768] tatcatgagaaatatccaactatctatcatctgcgaaaaaaattggtagattctactgataaagcggatttgcgcttaatctatttggccttagcgcata

[0769] tgattaagtttcgtggtcattttttgattgagggagatttaaatcctgataatagtgatgtggacaaactatttatccagttggtacaaacctacaatcaa

[0770] ttatttgaagaaaaccctattaacgcaagtggagtagatgctaaagcgattctttctgcacgattgagtaaatcaagacgattagaaaatctcattgc

[0771] tcagctccccggtgagaagaaaaatggcttatttgggaatctcattgctttgtcattgggtttgacccctaattttaaatcaaattttgatttggcagaa

[0772] gatgctaaattacagctttcaaaagatacttacgatgatgatttagataatttattggcgcaaattggagatcaatatgctgatttgtttttggcagctaa

[0773] gaatttatcagatgctattttactttcagatatcctaagagtaaatactgaaataactaaggctcccctatcagcttcaatgattaaacgctacgatgaa

[0774] catcatcaagacttgactcttttaaaagctttagttcgacaacaacttccagaaaagtataaagaaatcttttttgatcaatcaaaaaacggatatgca

[0775] ggttatattgatgggggagctagccaagaagaattttataaatttatcaaaccaattttagaaaaaatggatggtactgaggaattattggtgaaact

[0776] aaatcgtgaagatttgctgcgcaagcaacggacctttgacaacggctctattccccatcaaattcacttgggtgagctgcatgctattttgagaaga

[0777] caagaagacttttatccatttttaaaagacaatcgtgagaagattgaaaaaatcttgacttttcgaattccttattatgttggtccattggcgcgtggca

[0778] atagtcgttttgcatggatgactcggaaatctgaagaaacaattaccccatggaattttgaagaagttgtcgataaaggtgcttcagctcaatcattta

[0779] ttgaacgcatgacaaactttgataaaaatcttccaaatgaaaaagtactaccaaaacatagtttgctttatgagtattttacggtttataacgaattgac

[0780] aaaggtcaaatatgttactgaaggaatgcgaaaaccagcatttctttcaggtgaacagaagaaagccattgttgatttactcttcaaaacaaatcga

[0781] aaagtaaccgttaagcaattaaaagaagattatttcaaaaaaatagaatgttttgatagtgttgaaatttcaggagttgaagatagatttaatgcttcat

[0782] taggtacctaccatgatttgctaaaaattattaaagataaagattttttggataatgaagaaaatgaagatatcttagaggatattgttttaacattgacc

[0783] ttatttgaagatagggagatgattgaggaaagacttaaaacatatgctcacctctttgatgataaggtgatgaaacagcttaaaacgtcgccgttatac

[0784] tggttggggacgtttgtctcgaaaattgattaatggtattagggataagcaatctggcaaaacaatattagattttttgaaatcagatggttttgccaat

[0785] cgcaattttatgcagctgatccatgatgatagtttgacatttaaagaagacattcaaaaagcacaagtgtctggacaaggcgatagtttacatgaac

[0786] atattgcaaatttaggctgtagccctgctattaaaaaaggtattttacagactgtaaaagttgttgaatgaattggtcaaagtaatggggcggcataag

[0787] ccagaaaatatcgttattgaaatggcacgtgaaaatcagacaactcaaaagggccagaaaaattcgcgagagcgtatgaaacgaatcgaagaa

[0788] ggtatcaaagaattaggaagtcagattcttaaagagcatcctgttgaaaatactcaattgcaaaatgaaaagctctatctctattatctccaaaatgg

[0789] aagagacatgtatgtggaccaagaattagatatatcgtttaagtgattatgatgtcgatGCTattgttccacaaagtttccttaaagacgattcaa

[0790] tagacaataaggtcttaacgcgttctgataaaaaatcgtggtaaatcggataacgttccaaggaagaaggtcaaaaaagatgaaaaaactattgga

[0791] gacaacttctaaacgccaagttaatcactcaacgtaagtttgataatttaacgaaagctgaacgtggaggtttgagtgaacttgataaagctggtttt

[0792] atcaaacgccaattggttgaaactcgccaaatcactaagcatgtggcacaaattttggatagtcgcatgaatactaaatacgatgaaaatgaaaa

[0793] cttattcgagaggttaaagtgattaccttaaaatctaaattagtttctgacttccgaaaagatttccaattctataaagtacgtgagattaacaattacca

[0794] tcatgcccatgatgcgtatctaaatgccgtcgttggaactgctttgattaagaaatatccaaaacttgaatcggagtttgtctatggtgattataaagtt

[0795] tatgatgttcgtaaaatgattgctaagtctgagcaagaaataggcaaagcaaccgcaaaatatttcttttactctaatatcatgaacttcttcaaaaca

[0796] gaaattacacttgcaaatggagagattcgcaaacgccctctaatcgaaactaatggggaaactggagaaattgtctgggataaagggcgagattt

[0797] tgccacagtgcgcaaagtattgtccatgccccaagtcaatattgtcaagaaaacagaagtacagacaggcggattctccaaggagtcaattttac

[0798] caaaaaagaaattcggacaagcttattgctcgtaaaaaagactgggatccaaaaaaatatggtggttttgatagtccaacggtagcttattcagtcct

[0799] agtggttgctaaggtggaaaaagggaaatcgaagaagttaaaatccgttaaagagttactagggatcacaattatggaaagaagttcctttgaaa

[0800] aaaatccgattgactttttagaagctaaaggatataaggaagttaaaaaagacttaatcattaaactacctaaatatagtctttttgagttagaaaacg

[0801] gtcgtaaacggatgctggctagtgccggagaattacaaaaaggaaatgagctggctctgccaagcaaatatgtgaattttttatatttagctagtca

[0802] ttatgaaaagttgaagggtagtccagaagataacgaacaaaaacaattgtttgtggagcagcataagcattatttagatgagattattgagcaaatc

[0803] agtgaattttctaagcgtgttattttagcagatgccaatttagataaagttcttagtgcatataacaaacatagagacaaaccaatacgtgaacaagc

[0804] agaaaatattattcatttatttacgttgacgaatcttggagctcccgctgcttttaaatattttgatacaacaattgatcgtaaacgatatacgtctacaaa

[0805] agaagttttagatgccactcttatccatcaatccatcactggtctttatgaaacacgcattgatttgagtcagctaggaggtgacAGCGGCGG

[0806] TTCTTCCGGCGGCTCCAGCGGTTCCGAAACTCCGGGTACCTCTGAATCTGCGACCCCGG

[0807] AATCTTCTGGCGGCAGCTCTGGCGGTAGCAGCACCCTGAACATCGAAGATGAATATCG

[0808] TCTGCACGAAACCTCTAAAGAGCCGGACGTTTCCCTGGGCAGCACCTGGCTGTCCGATT

[0809] TCCCGCAGGCGTGGGCGGAAACCGGCGGCATGGGTCTGGCGGTTCGCCAGGCGCCGCT

[0810] GATCATCCCGCTGAAAGCGACCTCTACCCCGGTAAGCATCAAACAGTATCCGATGAGC

[0811] CAGGAAGCGCGTTTAGGTATCAAACCGCATATTCAGCGCCTGCTGGATCAGGGTATTC

[0812] TGGTGCCGTGCCAGTCTCCGTGGAACACTCCACTGCTTCCGGTCAAAAAACCGGGTAC

[0813] CAACGACTACCGTCCGGTACAGGATCTGCGTGAAGTTAACAAACGTGTTGAAGATATC

[0814] CATCCGACCGTACCGAACCCGTATAACCTGCTGTCTGGCCTGCCGCCGAGCCACCAGT

[0815] GGTACACCGTGCTGGATTTGAAAGATGCGTTTTTCTGTCTGCGCCTGCATCCGACCTCC

[0816] CAACCGCTGTTCGCCTTCGAATGGCGTGATCCGGAGATGGGTATTAGCGGCCAGCTGA

[0817] CTTGGACTCGTCTGCCGCAGGGCTTCAAAAACAGCCCGACGCTCTTCAACGAAGCGCT

[0818] GCATCGTGACCTGGCCGATTTCCGTATCCAGCACCCGGATCTGATCTTGCTGCAGTACG

[0819] TGGACGACCTGCTGCTGGCGGCAACGTCGGAACTGGACTGTCAGCAGGGCACTCGTGC

[0820] ACTGCTGCAGACCCTGGGTAACCTGGGTTATCGTGCATCTGCTAAAAAAGCCCAGATC

[0821] TGTCAGAAACAGGTTAAATACCTCGGCTACTTGCTGAAAGAAGGCCAGCGCTGGCTTA

[0822] CCGAAGCTCGTAAAGAGACTGTGATGGGTCAGCCGACGCCGAAAACCCCGCGCCAACT

[0823] GCGTGAATTCCTGGGTAAAGCTGGCTTCTGCCGTCTGTTCATTCCGGGCTTTGCTGAAA

[0824] TGGCGGCTCCGCTGTATCCACTGACCAAACCGGGTACCCTGTTCAACTGGGGCCCTGAC

[0825] CAGCAGAAAGCATACCAGGAAATTAAACAGGCTCTTCTGACCGCGCCGGCACTGGGCC

[0826] TGCCGGATCTGACCAAACCGTTCGAACTGTTTGTTGATGAAAAACAGGGTTACGCGAA

[0827] AGGCGTCCTGACCCAGAAACTGGGTCCGTGGCGCCGCCCGGTGGCGTACCTGAGCAAA

[0828] AAACTGGATCCGGTTGCAGCAGGTTGGCCGCCGTGCCTGCGCATGGTTGCTGCGATTG

[0829] CTGTGTTGACCAAAGATGCTGGTAAACTGACCATGGGTCAGCCGCTGGTGATCCTGGC

[0830] TCCGCACGCTGTTGAAGCGCTGGTAAAACAGCCGCCGGACCGCTGGTTGTCTAACGCC

[0831] CGTATGACCCACTATCAGGCTCTGCTGCTGGACACCGACCGTGTTCAGTTCGGCCCAGT

[0832] GGTGGCACTGAATCCAGCGACCCTGCTGCCACTGCCGGAAGAGGGTCTGCAGCACAAC

[0833] TGCCTTGATATCCTGGCCGAAGCGCACGGTACCCGTCCGGACTTAACCGATCAGCCGCT

[0834] GCCGGATGCTGATCATACCTGGTATACCGACGGTCCTCTTTGTTGCAGGAAGGTCAGC

[0835] GTAAAGCGGGTGCGGCCGTTACCACCGAAACTGAAGTAATTTGGGCCAAGGCGCTCCC

[0836] GGCGGGCACGTCCGCGCAGCGCGCTGAACTCATCGCACTGACCCAGGCGTTGAAAATG

[0837] GCAGAAGGCAAAAAACTGAATGTGTACACTGATTCCCGCTACGCGTTCGCAACCGCTC

[0838] ACATCCACGGCGAAATCTACCGCCGTCGCGGCTGGCTGACTAGCGAAGGTAAAGAAAT

[0839] TAAAAATAAAGATGAAATCCTGGCACTGCTGAAAGCACTGTTCCTGCCGAAACGTCTG

[0840] TCTATCATTCACTGCCCAGGCCACCAGAAGGGCCACTCCGCTGAAGCACGTGGTAACC

[0841] GCATGGCTGATCAGGCAGCACGTAAAGCGGCAATTACCGAAACCCCGGACACCTCTAC

[0842] CCTGCTGATCGAAAACTCTTCGCCGAGCGGTGGCTCTAAACGTACCGCTGACGGCTCCGAATTTGAATAA-3'

[0843] Amplification of SpCas9_M-MLVmut fragment 5' primer sequence (SEQ ID NO.32):

[0844] 5'-taaggaggttataaaaaatggataagaaatactcaataggcttagatatc-3'

[0845] Amplification of SpCas9_M-MLVmut fragment 3' primer sequence (SEQ ID NO.33):

[0846] 5'-agcgtttgcgacTTGAGATCGTTTTGGTCTGCGCG-3'

[0847] Amplification of the CbR resistance gene fragment 5' primer sequence (SEQ ID NO.34):

[0848] 5'-AAACGATCTCAAgtcgcaaacgctgtttgctc-3'

[0849] Amplification of the 3' primer sequence of the CbR resistance gene fragment (SEQ ID NO.35):

[0850] 5'-atccattttttataacctccttagagctcgaattc-3'

[0851] The three DNA fragments mentioned above were amplified separately using Takara's PrimeSTAR HS DNA Polymerase. The reaction system was: 34.4 μL ddH2O, 4 μL dNTP Mixture (2.5 mM each), 10 μL 5× Primestar Buffer, 0.3 μL 5'-Primer (50 μM), 0.3 μL 3'-Primer (50 μM), 0.5 μL template DNA (100 ng / μL), and 0.5 μL Primestar HS DNA Polymerase.

[0852] After preparing the PCR reaction system, polymerase chain reaction (PCR) was performed using the following cycle: 98°C for 30 seconds; 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 7 minutes, for a total of 30 cycles; and finally 72°C for 10 minutes. PCR products were recovered using the SanPrep column-based PCR product purification kit from Sangon Biotech (Shanghai) Co., Ltd. Specific steps for PCR product purification were followed according to the kit's instruction manual.

[0853] The two DNA fragments obtained were assembled using Gibson ligation. The specific reaction system was: 10 μL Gibson Assembly Master Mix (2X), 2.5 μL SpCas9_M-MLVmut fragment, 2.5 μL Apr fragment, 5 μL ddH2O. Incubate at 50°C for 15 minutes to obtain the KP_PE plasmid. Subsequently, 20 μL of the reaction product was transformed into the competent TOP10 strain of Escherichia coli and spread on an LB solid culture plate containing 50 μg / mL Apr. After the transformation liquid was absorbed by the solid culture plate, it was inverted and cultured in a 37°C incubator overnight. The transformed strain grown on the culture plate was transferred and preserved, and the plasmid was extracted using the SanPrep column-type plasmid DNA small-scale extraction kit of Sangon Biotech (Shanghai) Co., Ltd. for subsequent experiments. The specific steps of plasmid extraction were carried out according to the kit operating manual. At the same time, the plasmid was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing confirmation.

[0854] Figure 4 Schematic diagram of the construction of the PE protein expression plasmid. The figure shows the Gibson ligation of the carbenicillin resistance gene and the fusion protein SpCas9_M-MLVmut gene.

[0855] Example 3 - Construction of epegRNA expression plasmid pSGAb-km in lead editing

[0856] The specific construction method of the epegRNA expression plasmid pSGAb-km is as follows:

[0857] The epegRNA is inserted into the empty plasmid pegRNA2_tevopreQ1 (SEQ ID NO.12) in a specific order consisting of pegRNA spacer, pegRNA 3'extension and pegRNA scaffold. The coding sequence of the screening resistance gene KanR is preferably as shown in SEQ ID NO.36 in the sequence table.

[0858] SEQ ID NO.12:

[0859]

[0860] taaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtt

[0861] tcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataa

[0862] atcagcatccatgttggaatttaatcgcggcctagagcaagacgtttcccgttgaatatggctcatactcttcctttttcaatattattgaagcatttatc

[0863] agggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctga

[0864] cgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttTTGACAGCTAGCTCAGTCCG

[0865] AGACGATCGCTACGTCTCAtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggt

[0866] gttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccataggattttaacattttgcgttgttccaaaag

[0867] ttatcaacagcctagaacgtcataggaagcgattacagacactttatagctatcagcatgggaacataagggcaggatgaaatatgggtcttaaa

[0868] acgcaaatggtgaggttttagaggtattttttgaagatgattaaggcggtttgtttttaaaatttttggcggctctcaggctgcttacattttaaccagttc

[0869] agtgaaaagttctttttcagcaaatttctgtttagcaccatagctaaaacttgcgtggaacatattaagaattgaccgaaatgacacaatctcaattata

[0870] ttttttttgaaaagttttctttatcaaatattttaaatcattgatttatatataagtatgcattcattttaataaataatctttatttaacaatgatttatctatattca

[0871] attgtttaattattcttactaatattatctctatatcaatattttttatttaaaaacatatgtttagtagtgcttttgattaaagtaccagagggagggagcag

[0872] agctgaatgggaaatactcaccctagagcgattcttaaaaatcaccctaaagtattcccattcgatgtaccgtcggtcggtcgctttcgcatcaggg

[0873] atgacatcactgtatcaagctgccactgttatgattacgattgatagcaccgcctgaacacgctcataaccgccaattaaatgactactcattgcgtc

[0874] cgctactctgttcagttcccctagtaatagcgtttttccgatgtgtgctagcgtcactgtacctcatcacccacacatggacagattgagttacagac

[0875] attggctaaatttttggggtctatgcttgacaaagcgagttaaaaccttagaatttaagacaggtacattaagcccctgtggtgaaatcattagcggt

[0876] cgtcaaaccttaatgctttcgattgccgatatgtcagtatcgaagatctgtaccccataaattacggtaaagccccaagcaattgcaaggggctttta

[0877] tcttttttaacaaaaaaaatttataaatcaggattttataacactaaataatccaaagtacatgagtaagtcatgaccactcctgcgatgtgtgtagcac

[0878] tctgagtatccgtattatatcagtgatgtcatatacaaccacataatgcggatgtatcactaactccctagtatctttctgtctgcctgtgcgccccatc

[0879] agtcgattatcaacaagtaaatctgtcttttcttcaattaaatcatcaatttcaatagctgctatagggttgcgttcttcaagataatcatagatatttctac

[0880] gatcttggctatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgg

[0881] gaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttt

[0882] tcccagtcacgacgttgtaaaacgacggccagtgaattgacgcgtattgggatggtaccgggccccccctcgaggtcgacggtatcgatacgg

[0883] catcagagcagattgtactgagagtgcaccataatcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgctgtctttgac

[0884] aacagatgttttcttgcctttgatgttcagcaggaagctaggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcatatagcttgtaatca

[0885] cgacattgtttcctttcgcttgaggtacagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttttaacacaaggccagttttgt

[0886] tcagcggcttgtatgggccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaatgccgtcaatcgtcatttttgatccgcg

[0887] ggagtcagtgaacagataccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgttactgtgttagatgcaatcagcggtttcatcact

[0888] tttttcagtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcagccgtgcgttttttatcgctttgcagaagtttttgactttctt

[0889] gacggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcgccttggtagccatcttcagttccagtgtttgcttcaaatactaag

[0890] tatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgtcgcctgagctgtagttgccttcatcgatgaactgctgtacattttgata

[0891] cgtttttccgtcaccgtcaaagattgatttataatcctctacaccgttgatgttcaaagagctgtctgatgctgatacgttaacttgtgcagttgtcagtg

[0892] tttgtttgccgtaatgtttaccggagaaatcagtgtagaataaacggatttttccgtcagatgtaaatgtggctgaacctgaccattcttgtgtttggtct

[0893] tttaggatagaatcatttgcatcgaatttgtcgctgtctttaaagacgcggccagcgtttttccagctgtcaatagaagtttcgccgactttttgataga

[0894] acatgtaaatcgatgtgtcatccgcatttttaggatctccggctaatgcaaagacgatgtggtagccgtgatagtttgcgacagtgccgtcagcgttt

[0895] tgtaatggccagctgtcccaaacgtccaggccttttgcagaagagatatttttaattgtggacgaatcgaactcaggaacttgatatttttcatttttttg

[0896] ctgttcagggatttgcagcatatcatggcgtgtaatatgggaaatgccgtatgtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttg

[0897] cgcctcctgccagcagtgcggtagtaaaggttaatactgttgcttgttttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttag

[0898] cggtctgcttcttccagccctcctgtttgaagatggcaagttagttacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatacactttgccctttacacattttaggtcttgcctgctttatcagtaacaaacccgcgcgatttacttttcgacctcattctattagac-3'

[0899] SEQ ID NO.36:

[0900] 5'-ttagaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatga -3'

[0901] First, select a DNA fragment 20bp behind a certain NGG (the PAM sequence recognized by SpCas9 in this example is NGG) sequence on the Escherichia coli target gene. In this embodiment, the target sequence can be as shown in SEQ ID NO.8 in the sequence table: 5'-ccccacgctttcgcaacttc-3' (these 20 bases are called pegRNA spacer, NGG is not included therein, N is any base among A, T, C, G, and the coding sequence here is selected according to the specific target gene). This step is characterized in that: in order for the spacer fragment to be inserted into the pegRNA2_tevopreQ1 plasmid, TAGT is added to the 5' end of the single-stranded DNA sequence, and GTTTT is added to the 3' end. At the same time, the anti-complement sequence of the spacer needs to be synthesized, and GCTCTAAAAC is added to the 5' end of the anti-complement sequence. In this embodiment, the 28bp pegRNA 3'extension sequence can be selected from the sequence shown in any one of SEQ ID NO.48, SEQ ID NO.49 or SEQ ID NO.50. This step is characterized by the addition of GGTGC to the 5' end of the single-stranded DNA sequence to enable insertion of the pegRNA 3' extension into the pegRNA2_tevopreQ1 plasmid. A complementary sequence is also synthesized, and CGCG is added to the 5' end of the complementary sequence. A pegRNA scaffold is selected. This step is characterized by the addition of AGAGC to the 5' end of the single-stranded DNA sequence to enable insertion of the pegRNA scaffold into the pegRNA2_tevopreQ1 plasmid. A complementary sequence is also synthesized, and GCACC is added to the 5' end of the complementary sequence.

[0902] The xylB pegRNA spacer primer sequences used in the experiment were designed as follows:

[0903] xylB_F: 5'-TAGTccccacgctttcgcaacttcGTTTT-3' (SEQ ID NO.37)

[0904] xylB_R: 5'-gctctaaaacgaagttgcgaaagcgtgggg-3' (SEQ ID NO.38)

[0905] The specific primer designs for the pegRNA extension selected in the experiment, including point mutation, insertion, and deletion editing, are as follows:

[0906] +1T-to-A_F:5'-GGTGCcctttgttacctgaTgttgcgaaagcgt-3'(SEQ ID NO.39)

[0907] +1T-to-A_R:5'-CGCGacgctttcgcaacAtcaggtaacaaagg-3'(SEQ ID NO.40)

[0908] +1TAA ins_F:5'-GGTGCcctttgttacctgaaTTAgttgcgaaagcgt-3'(SEQ ID NO.41)

[0909] +1TAA ins_R:5'-CGCGacgctttcgcaacTAAttcaggtaacaaagg-3'(SEQ ID NO.42)

[0910] +1T del_F:5'-GGTGCcctttgttacctgagttgcgaaagcgt-3'(SEQ ID NO.43)

[0911] +1T del_R:5'-CGCGacgctttcgcaactcaggtaacaaagg-3'(SEQ ID NO.44)

[0912] The DNA sequence of the pegRNA scaffold (SEQ ID NO. 45) selected in the experiment is: 5'-tagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtc-3'. The specific primer sequences are designed as follows:

[0913] 5'-AGAGCtagaaatagcaagttaaaat-3'(SEQ ID NO.46)

[0914] 5'-GCACCgactcggtgccactttttca-3'(SEQ ID NO.47)

[0915] The above primers were synthesized by conventional methods at Sangon Biotech (Shanghai) Co., Ltd.

[0916] The DNA coding sequence of pegRNA 3'extension-point-mutation is shown as SEQ ID NO.48 in the sequence listing; the DNA coding sequence of pegRNA 3'extension-insertion is shown as SEQ ID NO.49 in the sequence listing; and the DNA coding sequence of pegRNA 3'extension-deletion is shown as SEQ ID NO.50 in the sequence listing.

[0917] SEQ ID NO.48:

[0918] 5'-cctttgttacctgaTgttgcgaaagcgt-3'

[0919] SEQ ID NO.49:

[0920] 5'-cctttgttacctgaaTTAgttgcgaaagcgt-3'

[0921] SEQ ID NO.50:

[0922] 5'-cctttgttacctgagttgcgaaagcgt-3'

[0923] The pegRNA2_tevopreQ1 plasmid was digested overnight with BsaI. The digestion system was: x μL 4000 ng pegRNA2_tevopreQ1, 2 μL BsaI-HF-v2, 5 μL 10× Cutsmart buffer, up to 50 μL ddH2O. Incubate at 37°C for 12 hours. Subsequently, the linearized plasmid was purified using the TIANquick Midi Purification Kit.

[0924] Each pair of designed and synthesized pegRNA spacer, pegRNA 3'extension, and pegRNA scaffold primers were annealed and phosphorylated. The specific reaction system and procedure are as follows:

[0925] The annealing reaction system was: 10 μL Primer_F (10 μM), 10 μL Primer_R (10 μM), 5 μL 5× Anneal Buffer. The annealing program was: 85°C for 3 min, 22°C for 10 s, ramp 0.1°C / s, and 12°C for 10 min.

[0926] The phosphorylation reaction system was as follows: 6 μL ddH2O, 2.5 μL oligonucleotide duplex, 1 μL 10× T4 DNA ligase buffer (NEB), and 0.5 μL T4 PNK (Takara). The reaction was carried out at 37°C for 30 min.

[0927] The DNA coding sequence of epegRNA-point-mutation is shown as SEQ ID NO.9 in the sequence listing; the DNA coding sequence of epegRNA-insertion is shown as SEQ ID NO.10 in the sequence listing; and the DNA coding sequence of epegRNA-deletion is shown as SEQ ID NO.11 in the sequence listing.

[0928] SEQ ID NO.9:

[0929] 5'-ccccacgctttcgcaacttcGTTTTAGAGCtagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggca ccgagtcGGTGCcctttgttacctgaTgttgcgaaagcgt-3'

[0930] SEQ ID NO.10:

[0931] 5'-ccccacgctttcgcaacttcGTTTTAGAGCtagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggca ccgagtcGGTGCcctttgttacctgaaTTAgttgcgaaagcgt-3'

[0932] SEQ ID NO.11:

[0933] 5'-ccccacgctttcgcaacttcGTTTTAGAGCtagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggca ccgagtcGGTGCcctttgttacctgagttgcgaaagcgt-3'

[0934] After completion of the above reaction, Golden Gate Assembly was performed. The GGA reaction system consisted of: 1 μL 10× T4 DNA ligase buffer, 20 ng digested pegRNA2_tevopreQ1, 1 μL annealed and phosphorylated pegRNA spacer, 1 μL annealed and phosphorylated pegRNA 3' extension, 1 μL annealed and phosphorylated pegRNA scaffold, 0.5 μL T4 DNA ligase, 0.5 μL BsaI-HF-v2, up to 10 μL ddH2O. The reaction procedure was as follows: 16 cycles of 37°C for 3 min, 16°C for 3 min, 37°C for 30 min, 70°C for 5 min, and 10°C forever.

[0935] The DNA coding sequence of the epegRNA expression plasmid pSGAb-km-point-mutation is shown in SEQ ID NO.5; the DNA coding sequence of the epegRNA expression plasmid pSGAb-km-insertion is shown in SEQ ID NO.6; and the DNA coding sequence of the epegRNA expression plasmid pSGAb-km-deletion is shown in SEQ ID NO.7.

[0936] SEQ ID NO.5:

[0937]

[0938] gaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagc

[0939] ggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaa

[0940] cgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagt

[0941] atatatgagtaaacttggtctgacagttagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaa

[0942] aaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaaca

[0943] tcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagtt

[0944] tatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcct

[0945] gagcgagTcgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaac

[0946] aatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccagggatcgcagtggtgagtaaccatgcatcatcaggagtacgga

[0947] taaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtt

[0948] tcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataa

[0949] atcagcatccatgttggaatttaatcgcggcctagagcaagacgtttcccgttgaatatggctcatactcttcctttttcaatattattgaagcatttatc

[0950] agggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctga

[0951] cgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttTTGACAGCTAGCTCAGTCCG

[0952] AGACGATCGCTACGTCTCAtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggt

[0953] gttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccataggattttaacattttgcgttgttccaaaag

[0954] ttatcaacagcctagaacgtcataggaagcgattacagacactttatagctatcagcatgggaacataagggcaggatgaaatatgggtcttaaa

[0955] acgcaaatggtgaggttttagaggtattttttgaagatgattaaggcggtttgtttttaaaatttttggcggctctcaggctgcttacattttaaccagttc

[0956] agtgaaaagttctttttcagcaaatttctgtttagcaccatagctaaaacttgcgtggaacatattaagaattgaccgaaatgacacaatctcaattata

[0957] ttttttttgaaaagttttctttatcaaatattttaaatcattgatttatatataagtatgcattcattttaataaataatctttatttaacaatgatttatctatattca

[0958] attgtttaattattcttactaatattatctctatatcaatattttttatttaaaaacatatgtttagtagtgcttttgattaaagtaccagagggagggagcag

[0959] agctgaatgggaaatactcaccctagagcgattcttaaaaatcaccctaaagtattcccattcgatgtaccgtcggtcggtcgctttcgcatcaggg

[0960] atgacatcactgtatcaagctgccactgttatgattacgattgatagcaccgcctgaacacgctcataaccgccaattaaatgactactcattgcgtc

[0961] cgctactctgttcagttcccctagtaatagcgtttttccgatgtgtgctagcgtcactgtacctcatcacccacacatggacagattgagttacagac

[0962] attggctaaatttttggggtctatgcttgacaaagcgagttaaaaccttagaatttaagacaggtacattaagcccctgtggtgaaatcattagcggt

[0963] cgtcaaaccttaatgctttcgattgccgatatgtcagtatcgaagatctgtaccccataaattacggtaaagccccaagcaattgcaaggggctttta

[0964] tcttttttaacaaaaaaaatttataaatcaggattttataacactaaataatccaaagtacatgagtaagtcatgaccactcctgcgatgtgtgtagcac

[0965] tctgagtatccgtattatatcagtgatgtcatatacaaccacataatgcggatgtatcactaactccctagtatctttctgtctgcctgtgcgccccatc

[0966] agtcgattatcaacaagtaaatctgtcttttcttcaattaaatcatcaatttcaatagctgctatagggttgcgttcttcaagataatcatagatatttctac

[0967] gatcttggctatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgg

[0968] gaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttt

[0969] tcccagtcacgacgttgtaaaacgacggccagtgaattgacgcgtattgggatggtaccgggccccccctcgaggtcgacggtatcgatacgg

[0970] catcagagcagattgtactgagagtgcaccataatcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgctgtctttgac

[0971] aacagatgttttcttgcctttgatgttcagcaggaagctaggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcatatagcttgtaatca

[0972] cgacattgtttcctttcgcttgaggtacagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttttaacacaaggccagttttgt

[0973] tcagcggcttgtatgggccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaatgccgtcaatcgtcatttttgatccgcg

[0974] ggagtcagtgaacagataccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgttactgtgttagatgcaatcagcggtttcatcact

[0975] tttttcagtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcagccgtgcgttttttatcgctttgcagaagtttttgactttctt

[0976] gacggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcgccttggtagccatcttcagttccagtgtttgcttcaaatactaag

[0977] tatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgtcgcctgagctgtagttgccttcatcgatgaactgctgtacattttgata

[0978] cgtttttccgtcaccgtcaaagattgatttataatcctctacaccgttgatgttcaaagagctgtctgatgctgatacgttaacttgtgcagttgtcagtg

[0979] tttgtttgccgtaatgtttaccggagaaatcagtgtagaataaacggatttttccgtcagatgtaaatgtggctgaacctgaccattcttgtgtttggtct

[0980] tttaggatagaatcatttgcatcgaatttgtcgctgtctttaaagacgcggccagcgtttttccagctgtcaatagaagtttcgccgactttttgataga

[0981] acatgtaaatcgatgtgtcatccgcatttttaggatctccggctaatgcaaagacgatgtggtagccgtgatagtttgcgacagtgccgtcagcgttt

[0982] tgtaatggccagctgtcccaaacgtccaggccttttgcagaagagatatttttaattgtggacgaatcgaactcaggaacttgatatttttcatttttttg

[0983] ctgttcagggatttgcagcatatcatggcgtgtaatatgggaaatgccgtatgtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttg

[0984] cgcctcctgccagcagtgcggtagtaaaggttaatactgttgcttgttttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttag

[0985] cggtctgcttcttccagccctcctgtttgaagatggcaagttagttacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatacactttgccctttacacattttaggtcttgcctgctttatcagtaacaaacccgcgcgatttacttttcgacctcattctattagac-3'

[0986] SEQ ID NO.6:

[0987]

[0988] aaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaaca

[0989] tcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagtt

[0990] tatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcct

[0991] gagcgagTcgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaac

[0992] aatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccagggatcgcagtggtgagtaaccatgcatcatcaggagtacgga

[0993] taaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtt

[0994] tcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataa

[0995] atcagcatccatgttggaatttaatcgcggcctagagcaagacgtttcccgttgaatatggctcatactcttcctttttcaatattattgaagcatttatc

[0996] agggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctga

[0997] cgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttTTGACAGCTAGCTCAGTCCG

[0998] AGACGATCGCTACGTCTCAtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggt

[0999] gttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccataggattttaacattttgcgttgttccaaaag

[1000] ttatcaacagcctagaacgtcataggaagcgattacagacactttatagctatcagcatgggaacataagggcaggatgaaatatgggtcttaaa

[1001] acgcaaatggtgaggttttagaggtattttttgaagatgattaaggcggtttgtttttaaaatttttggcggctctcaggctgcttacattttaaccagttc

[1002] agtgaaaagttctttttcagcaaatttctgtttagcaccatagctaaaacttgcgtggaacatattaagaattgaccgaaatgacacaatctcaattata

[1003] ttttttttgaaaagttttctttatcaaatattttaaatcattgatttatatataagtatgcattcattttaataattaatctttatttaacaatgatttatctatattca

[1004] attgtttaattattcttactaatattatctctatatcaatattttttatttaaaaacatatgtttagtagtgcttttgattaaagtaccagagggagggagcag

[1005] agctgaatgggaaatactcaccctagagcgattcttaaaaatcaccctaaagtattcccattcgatgtaccgtcggtcggtcgctttcgcatcaggg

[1006] atgacatcactgtatcaagctgccactgttatgattacgattgatagcaccgcctgaacacgctcataaccgccaattaaatgactactcattgcgtc

[1007] cgctactctgttcagttcccctagtaatagcgtttttccgatgtgtgctagcgtcactgtacctcatcacccacacatggacagattgagttacagac

[1008] attggctaaatttttggggtctatgcttgacaaagcgagttaaaaccttagaatttaagacaggtacattaagcccctgtggtgaaatcattagcggt

[1009] cgtcaaaccttaatgctttcgattgccgatatgtcagtatcgaagatctgtaccccataaattacggtaaagccccaagcaattgcaaggggctttta

[1010] tcttttttaacaaaaaaaatttataaatcaggattttataacactaaataatccaaagtacatgagtaagtcatgaccactcctgcgatgtgtgtagcac

[1011] tctgagtatccgtattatatcagtgatgtcatatacaaccacataatgcggatgtatcactaactccctagtatctttctgtctgcctgtgcgccccatc

[1012] agtcgattatcaacaagtaaatctgtcttttcttcaattaaatcatcaatttcaatagctgctatagggttgcgttcttcaagataatcatagatatttctac

[1013] gatcttggctatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgg

[1014] gaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttt

[1015] tcccagtcacgacgttgtaaaacgacggccagtgaattgacgcgtattgggatggtaccgggccccccctcgaggtcgacggtatcgatacgg

[1016] catcagagcagattgtactgagagtgcaccataatcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgctgtctttgac

[1017] aacagatgttttcttgcctttgatgttcagcaggaagctaggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcatatagcttgtaatca

[1018] cgacattgtttcctttcgcttgaggtacagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttttaacacaaggccagttttgt

[1019] tcagcggcttgtatgggccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaatgccgtcaatcgtcatttttgatccgcg

[1020] ggagtcagtgaacagataccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgttactgtgttagatgcaatcagcggtttcatcact

[1021] tttttcagtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcagccgtgcgttttttatcgctttgcagaagtttttgactttctt

[1022] gacggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcgccttggtagccatcttcagttccagtgtttgcttcaaatactaag

[1023] tatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgtcgcctgagctgtagttgccttcatcgatgaactgctgtacattttgata

[1024] cgtttttccgtcaccgtcaaagattgatttataatcctctacaccgttgatgttcaaagagctgtctgatgctgatacgttaacttgtgcagttgtcagtg

[1025] tttgtttgccgtaatgtttaccggagaaatcagtgtagaataaacggatttttccgtcagatgtaaatgtggctgaacctgaccattcttgtgtttggtct

[1026] tttaggatagaatcatttgcatcgaatttgtcgctgtctttaaagacgcggccagcgtttttccagctgtcaatagaagtttcgccgactttttgataga

[1027] acatgtaaatcgatgtgtcatccgcatttttaggatctccggctaatgcaaagacgatgtggtagccgtgatagtttgcgacagtgccgtcagcgttt

[1028] tgtaatggccagctgtcccaaacgtccaggccttttgcagaagagatatttttaattgtggacgaatcgaactcaggaacttgatatttttcatttttttg

[1029] ctgttcagggatttgcagcatatcatggcgtgtaatatgggaaatgccgtatgtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttg

[1030] cgcctcctgccagcagtgcggtagtaaaggttaatactgttgcttgttttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttag

[1031] cggtctgcttcttccagccctcctgtttgaagatggcaagttagttacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatacactttgccctttacacattttaggtcttgcctgctttatcagtaacaaacccgcgcgatttacttttcgacctcattctattagac-3'

[1032] SEQ ID NO.7:

[1033]

[1034] aatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccagggatcgcagtggtgagtaaccatgcatcatcaggagtacgga

[1035] taaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtt

[1036] tcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataa

[1037] atcagcatccatgttggaatttaatcgcggcctagagcaagacgtttcccgttgaatatggctcatactcttcctttttcaatattattgaagcatttatc

[1038] agggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctga

[1039] cgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttTTGACAGCTAGCTCAGTCCG

[1040] AGACGATCGCTACGTCTCAtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggt

[1041] gttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccataggattttaacattttgcgttgttccaaaag

[1042] ttatcaacagcctagaacgtcataggaagcgattacagacactttatagctatcagcatgggaacataagggcaggatgaaatatgggtcttaaa

[1043] acgcaaatggtgaggttttagaggtattttttgaagatgattaaggcggtttgtttttaaaatttttggcggctctcaggctgcttacattttaaccagttc

[1044] agtgaaaagttctttttcagcaaatttctgtttagcaccatagctaaaacttgcgtggaacatattaagaattgaccgaaatgacacaatctcaattata

[1045] ttttttttgaaaagttttctttatcaaatattttaaatcattgatttatatataagtatgcattcattttaataaataatctttatttaacaatgatttatctatattca

[1046] attgtttaattattcttactaatattatctctatatcaatattttttatttaaaaacatatgtttagtagtgcttttgattaaagtaccagagggagggagcag

[1047] agctgaatgggaaatactcaccctagagcgattcttaaaaatcaccctaaagtattcccattcgatgtaccgtcggtcggtcgctttcgcatcaggg

[1048] atgacatcactgtatcaagctgccactgttatgattacgattgatagcaccgcctgaacacgctcataaccgccaattaaatgactactcattgcgtc

[1049] cgctactctgttcagttcccctagtaatagcgtttttccgatgtgtgctagcgtcactgtacctcatcacccacacatggacagattgagttacagac

[1050] attggctaaatttttggggtctatgcttgacaaagcgagttaaaaccttagaatttaagacaggtacattaagcccctgtggtgaaatcattagcggt

[1051] cgtcaaaccttaatgctttcgattgccgatatgtcagtatcgaagatctgtaccccataaattacggtaaagccccaagcaattgcaaggggctttta

[1052] tcttttttaacaaaaaaaatttataaatcaggattttataacactaaataatccaaagtacatgagtaagtcatgaccactcctgcgatgtgtgtagcac

[1053] tctgagtatccgtattatatcagtgatgtcatatacaaccacataatgcggatgtatcactaactccctagtatctttctgtctgcctgtgcgccccatc

[1054] agtcgattatcaacaagtaaatctgtcttttcttcaattaaatcatcaatttcaatagctgctatagggttgcgttcttcaagataatcatagatatttctac

[1055] gatcttggctatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgg

[1056] gaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttt

[1057] tcccagtcacgacgttgtaaaacgacggccagtgaattgacgcgtattgggatggtaccgggccccccctcgaggtcgacggtatcgatacgg

[1058] catcagagcagattgtactgagagtgcaccataatcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgctgtctttgac

[1059] aacagatgttttcttgcctttgatgttcagcaggaagctaggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcatatagcttgtaatca

[1060] cgacattgtttcctttcgcttgaggtacagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttttaacacaaggccagttttgt

[1061] tcagcggcttgtatgggccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaatgccgtcaatcgtcatttttgatccgcg

[1062] ggagtcagtgaacagataccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgttactgtgttagatgcaatcagcggtttcatcact

[1063] tttttcagtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcagccgtgcgttttttatcgctttgcagaagtttttgactttctt

[1064] gacggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcgccttggtagccatcttcagttccagtgtttgcttcaaatactaag

[1065] tatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgtcgcctgagctgtagttgccttcatcgatgaactgctgtacattttgata

[1066] cgtttttccgtcaccgtcaaagattgatttataatcctctacaccgttgatgttcaaagagctgtctgatgctgatacgttaacttgtgcagttgtcagtg

[1067] tttgtttgccgtaatgtttaccggagaaatcagtgtagaataaacggatttttccgtcagatgtaaatgtggctgaacctgaccattcttgtgtttggtct

[1068] tttaggatagaatcatttgcatcgaatttgtcgctgtctttaaagacgcggccagcgtttttccagctgtcaatagaagtttcgccgactttttgataga

[1069] acatgtaaatcgatgtgtcatccgcatttttaggatctccggctaatgcaaagacgatgtggtagccgtgatagtttgcgacagtgccgtcagcgttt

[1070] tgtaatggccagctgtcccaaacgtccaggccttttgcagaagagatatttttaattgtggacgaatcgaactcaggaacttgatatttttcatttttttg

[1071] ctgttcagggatttgcagcatatcatggcgtgtaatatgggaaatgccgtatgtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttg

[1072] cgcctcctgccagcagtgcggtagtaaaggttaatactgttgcttgttttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttag

[1073] cggtctgcttcttccagccctcctgtttgaagatggcaagttagttacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatacactttgccctttacacattttaggtcttgcctgctttatcagtaacaaacccgcgcgatttacttttcgacctcattctattatagac-3'

[1074] Transform 5 μL of the resulting GGA product into E. coli TOP10 cells and plate them in TB medium supplemented with 50 μg / mL kanamycin. After the transformation solution has been absorbed by the solid culture plate, incubate the cells in an inverted position at 37°C overnight. Transformants grown on the culture plate were transferred and stored, and plasmids were extracted using the SanPrep Column-Based Plasmid DNA Miniprep Kit from Sangon Biotech (Shanghai) Co., Ltd. for subsequent experiments. Specific plasmid extraction procedures were followed according to the kit's instruction manual. The plasmids were then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing confirmation.

[1075] Figure 5 Schematic diagram of the construction of the epegRNA expression plasmid. The figure shows the insertion of the pegRNA spacer, pegRNA 3' extension, and pegRNA scaffold into the empty plasmid pegRNA2_tevopreQ1 in a specific order via Golden Gate ligation.

[1076] Example 4 - Preparation of Escherichia coli MG1655-i electroporation competent cells containing the BacPE system

[1077] After taking E.coli MG1655-i (containing pBbS8c-ddCpf1 plasmid and SpPE plasmid) out of the refrigerator, grow it on TB agar medium for 1 day at 30°C. Subsequently, single clone colonies were selected and grown into TB medium and grown to the stationary phase. 1 mL of bacterial solution was added to 100 mL of TB medium (containing Cb, Cm, arabinose and IPTG) and cultured in a shaker for 4 to 6 hours until OD600 = 0.5-0.6. After cooling the bacterial solution on ice for 20 minutes, centrifuge it at 4000 r·min-1 and wash it three times with 10% glycerol. Finally, resuspend the bacteria in 1 mL of 10% glycerol. During the electroporation experiment, 100 ng of epegRNA plasmid was added to 100 μL of competent cells, mixed well, and electroporated using a 2 mm electroporation cup. TB medium was added immediately after electroporation and the cells were revived in a shaker at 30°C for 1 hour. The bacteria were plated on TB agar medium supplemented with Cb, Cm, Km, arabinose, and IPTG, and then grown in a 30°C incubator for 36 hours.

[1078] Parameters used in all electroporation experiments: 200Ω, 2500V, 25μF.

[1079] Figure 6 The diagram below shows the construction and culture of E. coli MG1655-i containing the pBbS8c-ddCpf1 plasmid, the SpPE plasmid, and the epegRNA plasmid. The pBbS8c-ddCpf1 and SpPE plasmids in Step 1 can also be introduced in two separate stages.

[1080] Example 5—Characterization of the Editing Efficiency of the BacPE Prime Editing System

[1081] Collect all the bacteria on the plate and extract the bacterial genome using a column-based bacterial genomic DNA extraction kit. Then, use NanoDrop to determine the genome concentration. Use primers containing barcodes to amplify the target fragment on the genome (first round PCR). PCR reaction system: 2×Phanta Max Master Mix (5μL), H2O (3.5μL), P5-Primer (10μM) (0.5μL), P7-Primer (10μM) (0.5μL), genomic DNA (0.5μL). Run the following program in a PCR instrument: 98℃ for 60s; 98℃ for 30s, 60℃ for 60s, 40 cycles; 72℃ for 60s. Subsequently, use primers containing indexes to label the first round PCR products (second round PCR). The PCR reaction system consisted of 2× Phanta Max Master Mix (25 μL), H₂O (20 μL), P5-Primer (10 μM) (2 μL), P7-Primer (10 μM) (2 μL), and diluted first-round PCR product (1 μL). The following cycle was run in a PCR instrument: 98°C for 60 s; 98°C for 15 s, 65°C for 30 s, 72°C for 30 s, 15 cycles; and 72°C for 60 s. Analysis was performed by electrophoresis on a 1% agarose gel and purification was performed using the TIANquick Midi Purification Kit. Sequencing was then performed using an Illumina HiSeq (PE150).

[1082] Data were split using barcodeSpliter. Amplicon sequences were aligned to the reference sequence using CRISPResso2 with the -q 30 parameter (Clement et al., 2019). For point mutation analysis, the following parameters were used: "--amplicon_seq," "--prime_editing_pegRNA_spacer_seq," "--prime_editing_pegRNA_extension_seq," "--prime_editing_nicking_guide_seq," and "--discard_indel_reads." For indel counts, all other parameters remained unchanged, with the "--discard_indel_reads" parameter replaced by "--ignore_substitutions." When analyzing insertions or deletions, the following parameters were used: "--amplicon_seq", "--prime_editing_pegRNA_spacer_seq", "--expected_hdr_amplicon_seq", "--prime_editing_nicking_guide_seq", and "--discard_indel_reads". When counting indels, the other parameters remained unchanged, and the "--discard_indel_reads" parameter was replaced by "--ignore_substitutions".

[1083] Replacement efficiency = percentage of [non-discarded reads containing the edit] / [number of total reads].

[1084] Insertion or deletion efficiency = percentage of [homology-directed repair aligned reads] / [number of total reads].

[1085] The efficiency of indels = percentage of [number of discarded reads] / [number of total reads].

[1086] Figure 7The editing efficiency of BacPE for different types of editing was characterized. (A) Substitution editing was performed in xylB of E. coli MG1655 using BacPE. The epegRNA cut site is marked with a black triangle; (B) Insertion editing was performed in xylB of E. coli MG1655 using BacPE; (C) Deletion editing was performed in xylB of E. coli MG1655 using BacPE. The results showed that the prime editing efficiency was high when point mutations occurred at positions +5 and +6; when insertion editing was performed at position +6, the prime editing efficiency was high; when deletion editing was performed, the efficiency was high for deletions between positions +1 and +7.

[1087] Example 6 - Application of the BacPE Primer Editing System in Klebsiella pneumoniae

[1088] CRISPRi was used to inhibit the transcriptional levels of key DNA repair genes xseA, sbcB, or exoX in strain K. pneumoniae 1.6366. RNA was extracted from the cultured K. pneumoniae 1.6366 and the key gene inhibition effect was verified by RT-qPCR. After confirming that the above repair genes were inhibited, the BacPE protein expression plasmid PEE1[blaZ-p15a-araC] and the epegRNA expression plasmid pSGAb-km were transferred to K. pneumoniae 1.6366 (ΔsbcBΔxseAΔexoX) and cultured, and the BacPE editing efficiency in K. pneumoniae was then characterized. For specific implementation procedures, refer to Examples 1 to 5.

[1089] Figure 8 Figure 2 shows the effects of sbcB, xseA, and exoX on prime editing efficiency in K. pneumoniae. As shown in the figure, knocking out these three genes significantly improves prime editing efficiency in K. pneumoniae, suggesting that K. pneumoniae possesses a DNA repair mechanism for prime editing intermediates similar to that in E. coli.

[1090] While the basic concepts have been described above, it will be apparent to those skilled in the art that the detailed disclosure is merely illustrative and does not limit this specification. Although not explicitly stated herein, various modifications, improvements, and revisions to this specification may be made by those skilled in the art. Such modifications, improvements, and revisions are suggested in this specification and remain within the spirit and scope of the exemplary embodiments of this specification.

[1091] This specification also uses specific terms to describe the embodiments of this specification. For example, "one embodiment," "an embodiment," and / or "some embodiments" refer to a feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "one embodiment," "an embodiment," or "an alternative embodiment" two or more times in different locations in this specification do not necessarily refer to the same embodiment. Furthermore, certain features, structures, or characteristics of one or more embodiments of this specification may be appropriately combined.

[1092] In some embodiments, numbers are used to describe the quantity of components and attributes. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise stated, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the description and claims are approximate values, which may change according to the required characteristics of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the settings of such numerical values ​​are as accurate as possible within the feasible range.

[1093] Finally, it should be understood that the embodiments described in this specification are intended only to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, by way of example and not limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly described and illustrated in this specification.

Claims

1. A CRISPRi expression construct, comprising a xseA4 crRNA encoding nucleotide sequence, a sbcB3 crRNA encoding nucleotide sequence, a sbcB6 crRNA encoding nucleotide sequence and an exoX4 crRNA encoding nucleotide sequence.

2. The CRISPRi expression construct according to claim 1, wherein The xseA4 crRNA encoding nucleotide sequence is shown in SEQ ID NO.15, the sbcB3 crRNA encoding nucleotide sequence is shown in SEQ ID NO.16, the sbcB6 crRNA encoding nucleotide sequence is shown in SEQ ID NO.17, and the exoX4 crRNA encoding nucleotide sequence is shown in SEQ ID NO.

18.

3. The CRISPRi expression construct according to claim 1, wherein The nucleotide sequence of the CRISPRi expression construct is shown in SEQ ID NO.

1.

4. Use of the CRISPRi expression construct as described in any one of claims 1 to 3 in gene editing.

5. The use according to claim 4, characterized in that The gene editing is selected from at least one of gene cutting, gene deletion, gene insertion, base substitution, point mutation, transcription inhibition, transcription activation or base editing; preferably, the gene editing is selected from gene deletion, gene insertion or point mutation.

6. An epegRNA, comprising a pegRNA spacer, a pegRNA 3'extension and a pegRNA scaffold.

7. The epegRNA according to claim 6, wherein The encoding nucleotide sequence of the epegRNA is selected from the sequence shown in any one of SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO.

11.

8. An epegRNA expression construct, characterized in that: The epegRNA expression construct contains the nucleotide sequence encoding the epegRNA as described in claim 6 or 7.

9. The epegRNA expression construct according to claim 8, wherein The epegRNA expression construct is obtained by inserting the coding nucleotide sequence of the epegRNA as described in claim 6 or 7 into the empty plasmid pegRNA2_tevopreQ1, and the nucleotide sequence of the pegRNA2_tevopreQ1 is shown in SEQ ID NO.

12.

10. The epegRNA expression construct according to claim 8, wherein The nucleotide sequence of the epegRNA expression construct is selected from any one of SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.

7.

11. A pilot editing system, characterized in that: include: xseA4 crRNA or its encoding gene, sbcB3 crRNA or its encoding gene, sbcB6 crRNA or its encoding gene, and exoX4 crRNA or its encoding gene; and / or, PE protein or its encoding gene; and / or, epegRNA or its encoding gene.

12. The lead editing system according to claim 11, characterized in that: The coding sequence of the xseA4 crRNA is SEQ ID NO.15, the coding sequence of the sbcB3 crRNA is SEQ ID NO.16, the coding sequence of the sbcB6 crRNA is SEQ ID NO.17, and the coding sequence of the exoX4 crRNA is SEQ ID NO.18; The amino acid sequence of the PE protein is shown in SEQ ID NO.3; The epegRNA includes a pegRNA spacer, a pegRNA 3'extension and a pegRNA scaffold.

13. The lead editing system according to claim 11, characterized in that: The coding sequence of the epegRNA is selected from any one of SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.

11. The encoding nucleotide sequence of the PE protein is shown in SEQ ID NO.

4.

14. The lead editing system according to claim 11, characterized in that: Comprising a construct comprising: The xseA4 crRNA or its encoding gene, sbcB3 crRNA or its encoding gene, sbcB6 crRNA or its encoding gene, and exoX4 crRNA or its encoding gene; and / or, the PE protein or its encoding gene; And / or, the epegRNA or its encoding gene.

15. The lead editing system according to claim 14, characterized in that: The construct comprises: a CRISPRi expression construct according to any one of claims 1 to 3; and / or, PE protein expression construct; And / or, the epegRNA expression construct according to any one of claims 8 to 10.

16. The lead editing system according to claim 11, characterized in that: The PE protein expression construct includes a Cas9 protein framework element, a replicon element and a resistance gene expression element; preferably, the PE protein expression construct includes a Cas9 protein framework element as shown in SEQ ID NO.31, a P15a replicon element as shown in SEQ ID NO.30 and a resistance gene expression element as shown in SEQ ID NO.

29.

17. The lead editing system according to claim 11, characterized in that: The amino acid sequence of the PE protein is shown in SEQ ID NO.3, and the coding sequence of the PE protein is shown in SEQ ID NO.

4.

18. The lead editing system according to claim 11, characterized in that: The nucleotide sequence of the PE protein expression construct is shown in SEQ ID NO.

2.

19. A lead editing method, comprising contacting the lead editing system according to any one of claims 11 to 18 with a target gene to perform gene editing.

20. The lead editing method according to claim 19, wherein: The efficiency of prime editing can be improved by inhibiting or knocking out the expression of sbcB, exoX, and xseA 3'→5' single-stranded DNA exonucleases.

21. The lead editing method according to claim 19, characterized in that: The gene editing is selected from at least one of gene cutting, gene deletion, gene insertion, base substitution, point mutation, transcription inhibition, transcription activation or base editing; preferably, the gene editing is selected from gene deletion, gene insertion or point mutation.

22. An engineered bacterium comprising the CRISPRi expression construct according to any one of claims 1 to 3.

23. The engineered bacteria according to claim 22, characterized in that: The engineered bacteria are prokaryotic microorganisms; preferably, the engineered bacteria are selected from any one of actinomycetes, Bacillus, Streptomyces, yeast, Klebsiella pneumoniae or Escherichia coli; More preferably, the engineered bacteria is selected from Escherichia coli or Klebsiella pneumoniae.

24. An engineered bacterium obtained by gene editing the engineered bacterium using the lead editing method described in any one of claims 19 to 21.

25. The engineered bacteria according to claim 24, characterized in that The engineered bacteria are prokaryotic microorganisms; preferably, the engineered bacteria are selected from any one of actinomycetes, Bacillus, Streptomyces, yeast, Klebsiella pneumoniae or Escherichia coli; More preferably, the engineered bacteria is selected from Escherichia coli or Klebsiella pneumoniae.

26. An engineering bacteria construction kit, comprising the lead editing system according to any one of claims 11 to 18.

27. Use of the lead editing system according to any one of claims 11 to 18 or the lead editing method according to any one of claims 19 to 21 in gene editing.

28. The use according to claim 27, characterized in that The gene editing is selected from at least one of gene cutting, gene deletion, gene insertion, base substitution, point mutation, transcription inhibition, transcription activation or base editing; preferably, the gene editing is selected from gene deletion, gene insertion or point mutation.

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