A fusion protein, mutagenic vector, in vivo directed evolution system of specified region gene and application thereof

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to a fusion protein, a mutagenesis vector, a directed in vivo evolution system for a specified gene region, and their applications. The fusion protein of this invention comprises, from the N-terminus to the C-terminus, a SaCas9n domain, a flexible linker peptide, and an mPolA domain; the amino acid sequence of the SaCas9n domain is shown in SEQ ID NO.3; the amino acid sequence of the flexible linker peptide is shown in SEQ ID NO.7; and the amino acid sequence of the mPolA domain is shown in SEQ ID NO.5. The fusion protein constructed by this invention possesses the ability to efficiently mutate a specified gene region, and extends the length of the mutated region from less than 50 bp to more than 230 bp, enabling random mutation of a large region.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a fusion protein, a mutagenesis vector, a gene-directed evolution system for a specified region in vivo, and their applications. Background Technology

[0002] Directed evolution is a technique that modifies target gene sequences according to researchers' wishes and needs. This technique mimics the natural evolutionary process of modifying the function of biomolecules such as proteins. Traditional directed evolution techniques involve repeatedly performing mutation, selection, and amplification in vitro, which is typically time-consuming and relies heavily on manual intervention. In vivo continuous evolution technology avoids these tedious tasks, enabling continuous mutation and selection, and accelerating the evolutionary process.

[0003] In recent years, researchers have developed the EvolvR technology using CRISPR-Cas9 to continuously mutate specific genes. EvolvR technology fuses the nCas9 protein with the DNA polymerase Pol A. After the gRNA targets the site, the nCas9 protein cleaves one strand of the DNA double helix, and then the DNA polymerase Pol A, with its high mutation rate, repairs the cleavage while introducing random mutations, enabling continuous random mutations at multiple target sites. However, the fusion proteins formed by the current fusion of nCas9 and Pol A have a narrow effective mutation window, and the mutation rate decreases rapidly with increasing cleavage distance, severely restricting the continuous evolution of long genes or multi-domain proteins. Summary of the Invention

[0004] To address the shortcomings of existing technologies where fusion proteins formed by fusing nCas9 protein with DNA polymerase Pol A have narrow effective mutation windows and rapidly decreasing mutation rates with increasing cleavage distance, severely restricting the continuous evolution of long genes or multi-domain proteins, this invention utilizes a smaller SaCas9 protein and an evolutionary system designed with error-prone DNA polymerase. It provides a fusion protein, a mutagenic vector, a directed in vivo evolution system for a specified gene region, and its applications, enabling mutations within a larger window in a designated region and providing a foundation for the evolution of protein structure and function. To achieve the above objectives, this invention employs the following technical solution.

[0005] The first objective of this invention is to provide a SaCas9n-mPolA fusion protein, wherein the SaCas9n-mPolA fusion protein is composed of a SaCas9n domain, a flexible linker peptide, and an mPolA domain sequentially linked from the N-terminus to the C-terminus.

[0006] The SaCas9n domain is derived from the Cas9 mutant SaCas9-KKH of Staphylococcus aureus, which contains the H580A mutation and retains only single-strand cleavage activity against the non-target strand. Its amino acid sequence is shown in SEQ ID NO.3.

[0007] The flexible linker peptide is 32 amino acids long, and its amino acid sequence is shown in SEQ ID NO.7.

[0008] The mPolA domain is derived from a mutant DNA polymerase I of Escherichia coli K-12 strain, containing three amino acid substitutions at D424A, I709N, and A759R, and its amino acid sequence is shown in SEQ ID NO.5.

[0009] This invention optimizes the fusion method of SaCas9n and mPolA by using a flexible linker peptide of 32 amino acids to connect the SaCas9n domain and the mPolA domain. This ensures both the spatial flexibility of the nicking enzyme and the polymerase and maintains their synergistic function. Simultaneously, the SaCas9-KKH mutant (containing the H580A mutation) from Staphylococcus aureus is selected as the nicking enzyme, whose ability to recognize NNNRRT PAM sequences significantly expands the targeting range. The mPolA domain uses E. coli K-12 mutant DNA polymerase I containing mutations at D424A, I709N, and A759R, which has a higher mutation rate and continuous synthesis capacity, thus effectively expanding the mutation window. This solves the technical defects of existing fusion proteins, such as rapid decrease in mutation rate with distance and narrow effective mutation window, and achieves efficient continuous directed evolution of long genes or multi-domain proteins.

[0010] Furthermore, the amino acid sequence of the mPolA domain is shown in SEQ ID NO.5. This mutant (mPolA domain) loses its 5' exonuclease activity and cannot perform base correction. The mutagenic module Pol is an E. coli DNA polymerase I (PolA) mutant, whose amino acid sequence is shown in SEQ ID NO.5. Compared to wild-type PolA, this mutant has a mutation at position 424 (aspartic acid mutated to alanine, D424A), position 709 (isoleucine mutated to asparagine, I709N), and position 759 (alanine mutated to arginine, A759R), thereby losing its 5' to 3' exonuclease activity.

[0011] Furthermore, the amino acid sequence of the SaCas9n-mPolA fusion protein is shown in SEQ ID NO.8.

[0012] A second objective of this invention is to provide a mutagenesis vector comprising a nucleotide sequence encoding the SaCas9n-mPolA fusion protein, a tetracycline resistance operon promoter (for inducing expression of the fusion protein), and a kanamycin resistance marker gene.

[0013] A third objective of this invention is to provide a directed in vivo evolution system for a gene in a specified region, comprising the mutagenesis vector and the target vector, wherein the mutagenesis vector and the target vector are two mutually compatible plasmids that can coexist stably in the same host cell.

[0014] The target vector comprises a pBAD promoter, a target sequence, an aadA resistance marker gene, and a GFP reporter gene. The target sequence comprises a PAM sequence recognized by the SaCas9n domain (SaCas9-KKH), and the nucleotide sequence of the PAM sequence is shown in SEQ ID NO.27.

[0015] This invention utilizes a miniaturized SaCas9 derived from Staphylococcus aureus instead of SpCas9 derived from Streptococcus pyogenes to construct a novel in vivo continuous evolution system with long-distance mutations. The in vivo directed evolution system for a specific gene region constructed in this invention possesses the ability to efficiently mutate designated gene regions, and extends the length of the mutation region from less than 50 bp to over 230 bp, enabling random mutations over large regions. This overcomes the limitation of the relatively narrow mutation window in the current EvolvR system, which remains a major constraint on the efficient evolution of long genes or multi-domain proteins.

[0016] Furthermore, the designated region gene directed evolution system is used to randomly mutate a designated region of the target gene within living cells.

[0017] As an improvement, the system employs a miniaturized SaCas9n nuclease (SaCas9n domain) for single-strand cleavage of genomic target DNA as a specific targeting element. This SaCas9n is fused with a low-fidelity DNA polymerase PolA mutant (mPolA domain) via a flexible linker to form a complete mutagenesis module. This mutagenesis module is expressed under tetracycline induction and, guided by sgRNA, is located in a designated region of the target gene for random mutation. The miniaturized SaCas9n (SaCas9n domain) facilitates the long-distance action of the PolA mutant enzyme, effectively extending the mutation distance.

[0018] Existing in vivo evolution techniques based on Cas9 and error-prone DNA polymerases are limited by the small size of the mutation region, making it difficult to efficiently mutate larger gene sequences. This invention uses a miniaturized SaCas9n (SaCas9n domain) to extend the effective mutation distance of this system from 50 bp to over 230 bp, enabling efficient long-distance continuous mutation of specified gene regions within cells such as E. coli, thus providing a foundation for targeted modification of gene function.

[0019] A third objective of this invention is to provide a nucleotide sequence encoding the SaCas9n-mPolA fusion protein.

[0020] A fourth object of the present invention is to provide a cell containing a gene-directed in vivo evolution system for the designated region.

[0021] The cells are selected from bacterial cells, fungal cells, plant cells, or animal cells.

[0022] The fifth objective of this invention is to provide a dual-plasmid host bacterium, characterized in that it comprises the mutagenic vector and the target vector, wherein the host bacterium is Escherichia coli TG1 strain.

[0023] The sixth object of the present invention is to provide a method for directed in vivo evolution of genes in a designated region, characterized by comprising the following steps: The mutagenic vector and target vector were co-transformed into the host bacteria to construct the dual-plasmid host bacteria, and positive clones were obtained by double screening with chloramphenicol and kanamycin.

[0024] Induced expression: Tetracycline was added to induce the expression of the SaCas9n-mPolA fusion protein. The SaCas9n-mPolA fusion protein binds to the target sequence under the guidance of guide RNA. The SaCas9n domain performs single-strand cleavage of the non-target strand, and the mPolA domain performs error-prone DNA synthesis at the cleavage site, introducing mutations.

[0025] Screening for evolutionary mutants: Through resistance screening, evolutionary mutants with restored or enhanced function of the aadA resistance marker gene are obtained.

[0026] Furthermore, the conditions for inducing the expression of the SaCas9n-mPolA fusion protein by adding tetracycline are as follows: The concentration of tetracycline was 4.5 ng / mL to 5.5 ng / mL, and the induction time was 4 to 16 hours.

[0027] The seventh object of the present invention is to provide a method for directed evolution of proteins, comprising the following steps: (1) Introduce the gene-directed evolution system of the specified region into the host cell.

[0028] (2) Inducing expression of the target cleavage module Cas and the mutagenesis module Pol, and randomly mutating the target gene in the target gene expression module Tar under the guidance of gRNA.

[0029] (3) Screening to obtain mutants with the target trait.

[0030] Furthermore, the induced expression in step (2) is achieved by adding tetracycline or its derivatives.

[0031] An eighth object of the present invention is to provide the use of the fusion protein, the gene-directed evolution system of the designated region, the nucleotide sequence, the mutagenesis vector, or the dual-plasmid host bacterium in the preparation of a product of directed evolution of a protein or a gene encoding the protein.

[0032] Furthermore, the products include enzyme preparations, biocatalysts, diagnostic reagents, and therapeutic proteins.

[0033] Enzyme preparations: Industrial enzymes whose catalytic activity, thermal stability, substrate specificity, or organic solvent tolerance are enhanced through directed evolution.

[0034] Among them, biocatalysts are modified enzymes used to synthesize biological reactions.

[0035] Diagnostic reagents: Highly sensitive and stable enzyme markers or detection enzymes.

[0036] Therapeutic proteins: optimized antibodies, vaccines, or recombinant protein drugs.

[0037] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a SaCas9n-mPolA fusion protein. The SaCas9n-mPolA fusion protein comprises, from the N-terminus to the C-terminus, a SaCas9n domain, a flexible linker peptide, and an mPolA domain. The SaCas9n domain is derived from the Cas9 mutant SaCas9-KKH of Staphylococcus aureus, containing the H580A mutation, retaining only single-strand cleavage activity against non-target strands, and its amino acid sequence is shown in SEQ ID NO.3. The flexible linker peptide is 32 amino acids long, and its amino acid sequence is shown in SEQ ID NO.7. The mPolA domain is derived from a mutant DNA polymerase I of Escherichia coli strain K-12, containing three amino acid substitutions: D424A, I709N, and A759R. This invention optimizes the fusion method of SaCas9n and mPolA by using a flexible linker peptide of 32 amino acids to connect the SaCas9n domain and the mPolA domain. This ensures both the spatial flexibility of the nicking enzyme and the polymerase and maintains their synergistic function. Simultaneously, the SaCas9-KKH mutant (containing the H580A mutation) from Staphylococcus aureus is selected as the nicking enzyme, whose ability to recognize NNNRRT PAM sequences significantly expands the targeting range. The mPolA domain uses E. coli K-12 mutant DNA polymerase I containing mutations at D424A, I709N, and A759R, which has a higher mutation rate and continuous synthesis capacity, thus effectively expanding the mutation window. This solves the technical defects of existing fusion proteins, such as rapid decrease in mutation rate with distance and narrow effective mutation window, and achieves efficient continuous directed evolution of long genes or multi-domain proteins.

[0038] 2. This invention uses a miniaturized SaCas9 derived from Staphylococcus aureus instead of SpCas9 derived from Streptococcus pyogenes to construct a novel in vivo continuous evolution system for long-distance mutation. The gene-directed evolution system for a specified region constructed in this invention has the ability to efficiently mutate a designated gene region, and extends the length of the mutation region from less than 50 bp to over 230 bp, enabling random mutations over large regions. This overcomes the limitation of the relatively narrow mutation window of the current EvolvR system, which remains a major constraint on the efficient evolution of long genes or multi-domain proteins. The main reason for the significant expansion of the mutation window in the gene-directed evolution system provided by this invention is the low binding stability of SaCas9n to target DNA, faster dissociation after cleavage, and higher tolerance to mismatches introduced by polymerase. This breaks the bottleneck of excessively strong binding of SpCas9n limiting low-fidelity PolA extension, thereby significantly expanding the effective mutation window to over 230 bp, achieving efficient random point mutations in a specified large region. Attached Figure Description

[0039] Figure 1 This is a basic functional pattern diagram of directed in vivo evolution of genes in a specified region in this invention; wherein: (A) is the sequence that the nuclease Cas9n protein targets and recognizes, and then cleaves single-stranded DNA; (B) is a fault-prone DNA polymerase mPolA that synthesizes DNA from the nick and enters the mutation process.

[0040] Figure 2 This is a plasmid map of a preferred module in this invention; wherein: (A) is a mutagenesis vector containing a tetracycline-induced expression promoter tetR / tetA, a targeted cleavage module SaCas9n-KKH, a flexible linker 32aa-linker, a mutagenesis module of error-prone DNA polymerase mPolA, and a gRNA expression cassette. (B) is the target gene expression plasmid containing the spectinomycin resistance gene aadA and green fluorescent protein GFP, with a gRNA recognition sequence at the front end. Seven early termination loss-of-function aadA mutants, including Q12, were constructed.

[0041] Figure 3 Modeling the structure of the CasPol fusion protein, which is the fusion protein of the targeting cleavage module Cas and the mutagenesis module Pol in this invention; wherein: (A) and (B) are the top two results for AlphaFold2 modeling, where the darker color represents the modeled structure and the lighter color represents the crystal structures of SaCas9 (PDB ID: 5AXW) and PolA (PDB ID: 1D8Y); the structure alignment was performed using TM-align and the protein structure was displayed using PyMOL.

[0042] Figure 4 This invention compares the mutagenic efficiency (number of colonies recovering spectinomycin resistance) of different compositions of the cleavage module and mutagenic module on the aadA gene carrying the Q12 mutation; wherein: (A) represents the uncutting module and the mutation module; (B) contains only the cutting module SaCas9n-KKH; (C) contains only the mutagenic module mPolA; (D) indicates the presence of both a cleavage module and a mutagenesis module; the left side shows no tetracycline induction; the right side shows induction with 5 ng / mL tetracycline.

[0043] Figure 5 To analyze the mutation rate of the aadA gene carrying the Q12 mutation based on the different compositions of the cleavage module and the mutagenesis module in this invention, the method used was to determine the mutation rate by using the Ma-Sandri-Sarka maximum likelihood estimator combined with the aadA functional recovery colony number.

[0044] Figure 6 This is the result of the green fluorescent protein expression analysis of positive colonies obtained by screening on plates containing 200 μg / mL spectinomycin after mutagenesis of the aadA gene carrying the Q12 mutation using the CasPol mutagenesis system in this invention; wherein: (A) is a white light channel image of colonies that have recovered resistance after mutagenesis; (B) Image of green fluorescent protein expression observed in the ultraviolet light channel of mutagenized colonies that have recovered resistance; (C) is an image of the original colony under ultraviolet light.

[0045] Figure 7 This is the sequencing analysis result of the aadA mutation sites in positive colonies obtained by screening on plates containing 200 μg / mL spectinomycin after mutagenesis of the aadA gene carrying the Q12 mutation using the CasPol mutagenesis system in this invention; wherein: (A) is the Sanger sequencing result of the aadA gene of the original unmutated colony, with the Q12 site being the stop codon TAA; (B) is the Sanger sequencing result of the aadA gene of colony 1 after mutagenesis and recovery of resistance. The Q12 site is the homozygous state of CAA, that is, TAA is almost completely mutated to CAA. (C) is the Sanger sequencing result of the aadA gene in colony 2 after mutagenesis and recovery of resistance. The Q12 site is a heterozygous state of TAA and CAA, with CAA being the majority. (D) is the Sanger sequencing result of the aadA gene in colony 3 after mutagenesis and recovery of resistance. The Q12 site is a heterozygous state of TAA and CAA, and the proportions of the two are close.

[0046] Figure 8 This invention describes the observation of the number of positive colonies obtained by screening on plates containing 200 μg / mL spectinomycin after mutagenesis of the aadA gene carrying six different mutations (excluding Q12) using the CasPol mutagenesis system; wherein: (A) Comparison of the number of spectinomycin-resistant colonies before and after mutagenesis at the R22 site; (B) Comparison of the number of spectinomycin-resistant colonies before and after mutagenesis at the R57 site; (C) Comparison of the number of spectinomycin-resistant colonies before and after mutagenesis at the R84 site; (D) Comparison of the number of spectinomycin-resistant colonies before and after mutagenesis at the E87 site; (E) Comparison of the number of spectinomycin-resistant colonies before and after mutagenesis at the Q108 site; (F) Comparison of the number of spectinomycin-resistant colonies before and after mutagenesis at the Q160 site; The left side shows the result without tetracycline induction; the right side shows the result with 5 ng / mL tetracycline induction.

[0047] Figure 9 This invention uses the CasPol mutagenesis system to analyze the mutation rate of the aadA gene (excluding the key active site E87) carrying six different mutations. The horizontal axis represents the distance of the mutation site from the gRNA recognition sequence. The mutation rate is represented by the error bar, which indicates the mean error (SD). Three independent samples were used, and a two-tailed t-test was employed. *p<0.05, **p<0.01, ***p<0.001. Detailed Implementation

[0048] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0049] The plasmids and strains used in the following examples are from the following sources: pEvolvR plasmid was purchased from addgene; pBAD33 plasmid was purchased from Wuhan Miaoling Biotechnology Co., Ltd.; Escherichia coli DH5α and Escherichia coli TG1 (strains) were purchased from Shanghai Beyotime Biotechnology.

[0050] The reagents and culture media involved in the following examples are as follows: Hieff Canace® Plus High-Fidelity DNA Polymerase was purchased from Yisheng Biotechnology (Shanghai); Pfu DNA polymerase, calcium chloride, spectinomycin, kanamycin, and chloramphenicol were purchased from Shanghai Sangon Biotech; 2×Taq PCR MasterMix and tetracycline were purchased from Beijing Solarbio Science & Technology. BamH I, EcoR I, Xba Restriction endonucleases I were purchased from NEB (China); T4 DNA Ligase, Seamless Cloning Kit, DNA Recovery Kit, Plasmid Extraction Kit, and One-Step Competent Bacterial Preparation Kit were purchased from Shanghai Beyotime Biotechnology.

[0051] LB solid medium consists of 0.5% (m / v) yeast extract, 1% (m / v) tryptone, 1% (m / v) sodium chloride, and 2% (m / v) agar powder.

[0052] Example 1: Design of the SaCas9n-mPolA (CasPol) fusion protein of the mutagenesis system This embodiment constructs a novel fusion protein, SaCas9n-mPolA (hereinafter referred to as CasPol), for targeting DNA mutagenesis. This fusion protein consists of the following three functional modules:

[0053] (1) Nuclease domain: derived from Staphylococcus aureus ( Staphylococcus aureus The Cas9 mutant SaCas9-KKH recognizes the PAM sequence 5'-NNGRRT-3' (SaCas9-KKH PAM, SEQ ID NO.27). Based on this, an H580A point mutation is further introduced, allowing it to retain cleavage activity only on the non-target strand (i.e., the top DNA strand), forming the cleavage enzyme form SaCas9n (1052 amino acids in total, as shown in SEQ ID NO.3).

[0054] (2) Error-prone DNA polymerase domain: A mutant DNA polymerase I (mPolA, as shown in SEQ ID NO.5) derived from Escherichia coli strain K-12 was used, containing three amino acid substitutions at D424A, I709N, and A759R, with a total length of 927 amino acids. This mutant combination significantly reduced its replication fidelity and endowed it with error-prone synthesis capability.

[0055] (3) Flexible linker peptide: A flexible peptide segment of 32 amino acids (Sequence 6: SGGSSGGSSGSETPGTSESATPESSGGSSGGS, as shown in SEQ ID NO.7) was used to fuse the C-terminus of SaCas9n-KKH with the N-terminus of mPolA to construct the complete CasPol fusion protein (as shown in SEQ ID NO.8). The CasPol fusion protein is abbreviated as CasPol or fusion protein. The mutagenic process of CasPol on the target gene is as follows... Figure 1 As shown.

[0056] To assess whether the fusion strategy affects the natural conformation and potential activity of each functional domain, this invention utilizes the AlphaFold2 artificial intelligence algorithm to perform high-precision modeling of the full-length three-dimensional structure of CasPol. Subsequently, the structural alignment tool TM-align is used to compare the SaCas9n-KKH region and mPolA region in the model with their corresponding known crystal structures, and TM-score values ​​are calculated to quantify structural similarity.

[0057] The results are as follows Figure 3As shown, the TM-score of the SaCas9n-KKH domain in CasPol compared to the wild-type SaCas9-KKH crystal structure is 0.98785; the TM-score of the mPolA domain compared to its native crystal structure is 0.94161. Both TM-scores are above 0.9, indicating that both functional modules in the fusion protein (i.e., the cleavage module SaCas9-KKH and the mutagenic module mPolA) highly retain their native folding conformations, possessing the structural basis for maintaining their respective biological activities.

[0058] Therefore, the CasPol fusion protein is structurally compatible with both targeted DNA binding / cleavage activity and error-prone DNA synthesis capability, providing a molecular basis for the subsequent development of precise and controllable targeted mutagenesis systems.

[0059] Example 2: Construction of a series of mutagenic vectors To construct a modular and controllable targeted mutagenesis system, this embodiment modifies the existing plasmid pEvolvR-enCas9-PolI3M-TBD (purchased from Addgene, catalog number 113077). First, using PCR amplification combined with seamless cloning technology, 1bp-7400bp and 9700bp-12753bp were deleted from the plasmid, retaining only the 7400bp-9700bp region, thus obtaining the basic backbone vector pCNACE. The backbone vector pCNACE is also known as the pCNACE vector.

[0060] Subsequently, Nanjing Genscript Biotech Co., Ltd. was commissioned to synthesize a gRNA expression cassette targeting the upstream region of the target gene (as shown in SEQ ID NO.1), with a spacer sequence of 5'-AGCAGCGGCATGAGGGAAGC-3' (as shown in SEQ ID NO.27). This gRNA expression cassette was inserted into the backbone vector pCNACE to construct a control vector pCNACE-tet that does not contain nuclease and polymerase coding sequences ("tet" indicates that the subsequent functional module is regulated by a tetracycline-inducible promoter). The control vector pCNACE-tet is also known as the pCNACE-tet vector.

[0061] Based on this, the following three functional carriers are constructed respectively: (1) Targeted cleavage module vector pCNACE-tet-Cas: The whole gene was synthesized with codon-optimized SaCas9n-KKH coding sequence of Escherichia coli DH5α strain (as shown in SEQ ID NO.2), and then... EcoR I and XbaThe enzyme was cleaved at a double restriction site and directionally cloned downstream of the tetracycline-inducible promoter (Ptet) in the pCNACE-tet vector to obtain the vector pCNACE-tet-Cas expressing the SaCas9n-KKH cleavage enzyme, also known as the targeted cleavage module vector pCNACE-tet-Cas. The targeted cleavage module vector pCNACE-tet-Cas is also referred to as the pCNACE-tet-Cas vector or the vector pCNACE-tet-Cas.

[0062] (2) Error-prone synthesis module vector pCNACE-tet-Pol: The coding sequence of the whole gene encoding the error-prone DNA polymerase mPolA (as shown in SEQ ID NO.4) was synthesized and inserted downstream of the Ptet promoter in the pCNACE-tet vector using seamless cloning technology to construct the vector pCNACE-tet-Pol that expresses only mPolA, i.e., the error-prone synthesis module vector pCNACE-tet-Pol. Among them, the error-prone synthesis module vector pCNACE-tet-Pol is also called pCNACE-tet-Pol vector or vector pCNACE-tet-Pol.

[0063] (3) Fusion mutagenesis module vector pCNACE-tet-CasPol: A DNA fragment encoding a 32-amino acid flexible linker peptide (linker fragment, as shown in SEQ ID NO. 6, with the corresponding amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS, as shown in SEQ ID NO. 7) was synthesized. The linker fragment and the coding sequence of mPolA were amplified using high-fidelity DNA polymerase, and the linker-mPolA fusion fragment was inserted into the 3' end of the SaCas9n-KKH coding sequence in the pCNACE-tet-Cas vector through one-step seamless cloning, enabling the expression of the SaCas9n-KKH–linker–mPolA fusion protein (i.e., CasPol fusion protein or CasPol mutagenesis system, abbreviated as CasPol, as shown in SEQ ID NO. 8) under the Ptet promoter. Finally, the bifunctional vector pCNACE-tet-CasPol, which simultaneously targets DNA cleavage sites and performs error-prone synthesis, was obtained. The fusion mutagenesis module carrier pCNACE-tet-CasPol is also known as the pCNACE-tet-CasPol carrier or carrier pCNACE-tet-CasPol.

[0064] See the structural diagrams of the above-mentioned carriers. Figure 2In (A), SaCas9n-KKH and mPolA in the CasPol fusion protein are linked by a 32aa flexible peptide segment, ensuring the spatial independence and synergistic effect of the two functional domains. Figure 3 ).

[0065] Example 3: Construction of the Mutagenic Target Vector To establish a reporter target that can be used to evaluate the activity of a mutagenesis system, this embodiment constructs a dual-label mutagenesis target vector pBAD33J-aadA-GFP. Details are as follows:

[0066] First, using pBAD33 plasmid as a template, PCR was performed to amplify only the 1318bp-5352bp region, yielding PCR product 1. Simultaneously, the constitutive promoter J23101 (a strong synthetic biology promoter, as shown in SEQ ID NO.27) was amplified from plasmid pEvolvR-enCas9-PolI3M-TBD, yielding PCR product 2. The two PCR products (PCR product 1 and PCR product 2) were then sequentially processed... BamH I and Kpn After double digestion with enzyme I, the DNA was ligated using T4 DNA ligase to construct the basic backbone vector pBAD33J. The basic backbone vector pBAD33J is simply referred to as the pBAD33J vector.

[0067] Subsequently, Nanjing GenScript Biotech Co., Ltd. was commissioned to synthesize the whole genome sequence encoding green fluorescent protein (GFP) (as shown in SEQ ID NO.9), and then... Pst I and Hind The III double restriction enzyme site was directionally inserted downstream of the J23101 promoter in the pBAD33J vector to obtain the intermediate vector pBAD33J-GFP expressing GFP. The intermediate vector pBAD33J-GFP expressing GFP is simply referred to as the pBAD33J-GFP vector.

[0068] To further introduce target sites that can be recognized by gRNA and achieve mutagenic phenotype screening, a whole-genome aadA gene fragment (as shown in SEQ ID NO.11) was synthesized. This fragment had a specific gRNA recognition sequence fused to its 5' end (the spacer sequence matched the gRNA expression cassette in Example 2), thus obtaining the aadA-spacer fragment. Using seamless cloning technology, this aadA-spacer fragment was precisely inserted upstream of the GFP coding sequence in the pBAD33J-GFP vector (i.e., located between the J23101 promoter and GFP), thereby constructing the final mutagenic target vector pBAD33J-aadA-GFP (see schematic diagram). Figure 2(B) refers to the mutagenic target vector. Among them, the mutagenic target vector pBAD33J-aadA-GFP is also known as the target vector.

[0069] In this target vector, the aadA gene serves as a mutagenic "target" embedded between the strong promoter and the reporter gene GFP. When the mutagenesis system introduces a frameshift or premature termination mutation into the aadA region, it may disrupt its interference with downstream GFP expression (e.g., through transcriptional readthrough or translational coupling mechanisms), thereby indirectly reflecting the mutagenesis efficiency through changes in fluorescence signals. Simultaneously, aadA itself encodes an aminoglycoside adenosyltransferase, conferring streptomycin / spectinomycin resistance, and can also be used for positive screening of functional mutants.

[0070] Example 4: Construction of aadA loss-of-function mutant in target vector To systematically evaluate the mutation efficiency and scope of the CasPol mutagenesis system at different sites of target genes in *E. coli*, this invention constructed a series of premature termination mutants of the aadA gene as loss-of-function reporter targets. Specifically, seven nonsense mutations were introduced into the aadA gene sequence (as shown in SEQ ID NO. 11), replacing specific codons with stop codons, thereby resulting in the expression of truncated and nonfunctional aadA protein. The constructed mutants include (with the original codons → stop codons and their corresponding sequence numbers in parentheses):

[0071] Q12 (CAA→TAA, as shown in SEQ ID NO.13).

[0072] R22 (CGC→TGA, as shown in SEQ ID NO.15).

[0073] R57 (AGG→TGA, as shown in SEQ ID NO.17).

[0074] R84 (CGC→TGA, as shown in SEQ ID NO.19).

[0075] E87 (GAA→TAA, as shown in SEQ ID NO.21).

[0076] Q108 (CAA→TAA, as shown in SEQ ID NO.23).

[0077] Q160 (CAG→TAA, as shown in SEQ ID NO.25).

[0078] The mutation sites mentioned above are 23bp, 53bp, 158bp, 239bp, 248bp, 311bp and 467bp from the 3' end of the gRNA recognition sequence (spacer) (as shown in SEQ ID NO.27), respectively, covering a wide region from the proximal end to the distal end, and are used to evaluate the spatial distribution characteristics of the mutagenic activity of the CasPol mutagenesis system.

[0079] For each mutant, site-directed mutagenesis PCR was used to amplify two fragments upstream and downstream of the aadA gene mutation site to ensure precise introduction of the mutation. Subsequently, using seamless cloning technology, these two fragments were inserted together upstream of the GFP coding sequence in the pBAD33J-GFP vector (i.e., between the J23101 promoter (SEQ ID NO.28) and GFP), replacing the original wild-type aadA gene sequence (as shown in SEQ ID NO.11).

[0080] This resulted in a series of mutant target vectors (mutant vectors), which were named as follows: pBAD33J-aadA_Q12-GFP.

[0081] pBAD33J-aadA_R22-GFP.

[0082] pBAD33J-aadA_R57-GFP.

[0083] pBAD33J-aadA_R84-GFP.

[0084] pBAD33J-aadA_E87-GFP.

[0085] pBAD33J-aadA_Q108-GFP.

[0086] pBAD33J-aadA_Q160-GFP.

[0087] All constructed mutant vectors retained intact gRNA targeting sites and lost spectinomycin / streptomycin resistance due to premature termination by aadA, serving as a basis for screening CasPol-mediated reversion mutations or functional recovery. These vectors provide a standardized genetic platform for subsequent quantitative analysis of CasPol mutagenesis efficiency at different distances (see structural diagram). Figure 2 (B) in the middle.

[0088] Example 5: Construction of a dual-plasmid Escherichia coli host containing a mutagen and a target vector To establish a dual-plasmid coexistence system that can be used to evaluate the mutagenic activity of the CasPol mutagenesis system, this embodiment sequentially introduces the target vector and the mutagenic vector into the *Escherichia coli* TG1 strain to construct a stable dual-plasmid host bacterium. The specific procedures are as follows:

[0089] (1) Activation and preparation of competent cells of Escherichia coli TG1 strain: The glycerol-preserved strain of Escherichia coli TG1 was streaked onto antibiotic-free LB solid medium and incubated upside down in a 37°C incubator for 16 hours to activate the strain. The next day, single colonies were picked and inoculated into 10 mL of antibiotic-free LB liquid medium and cultured at 37°C with shaking until OD. 600 The concentration reached 0.3, and chemocompetent cells were prepared using the CaCl2 method to obtain TG1 competent cells.

[0090] (2) Transformation and functional verification of the target vectors: The mutagenic target vectors constructed in Example 3 and the various mutant target vectors constructed in Example 4 (including wild-type pBAD33J-aadA-GFP and its series of premature termination mutant vectors) were transformed into the above-mentioned TG1 competent cells. The transformation products were plated on LB solid medium containing 50 μg / mL chloramphenicol (Cm) and cultured at 37°C for 12 hours to screen for positive clones. Then, a single colony was picked and inoculated into 2 mL of LB liquid medium containing 50 μg / mL Cm and cultured at 37°C with shaking for 12 hours. 1 mL of culture was taken (adjusted OD) 600 ≈1) Spread the sample on LB solid medium containing 50 μg / mL chloramphenicol + 200 μg / mL spectinomycin (Spc) and incubate at 37°C for 16 hours to verify the functional status of the aadA gene: the pBAD33J-aadA-GFP vector should be able to grow on Spc plates (with spectinomycin resistance); premature termination mutants (such as the six mutant target vectors Q12, R22, R57, R84, E87, Q108, and Q160) should not be able to grow (loss of function), thus confirming that the target design is correct.

[0091] (3) Construction and preservation of dual-plasmid host bacteria: TG1 monoclonal strains containing the target vector that have been functionally verified and meet expectations were selected, and competent cells were re-prepared. Subsequently, various mutagenic vectors constructed in Example 2 (such as pCNACE-tet-Cas, pCNACE-tet-Pol, and pCNACE-tet-CasPol) were transformed into these competent cells. The transformation products were plated on LB solid medium containing 50 μg / mL chloramphenicol + 50 μg / mL kanamycin (Kan) (Note: the pCNACE series of mutagenic vectors carries a kanamycin resistance marker), and cultured at 37°C for 16 hours to screen for dual-plasmid positive clones carrying both the target vector and the mutagenic vector. Single colonies were picked and inoculated into 2 mL of double-antibody (Cm+Kan) LB liquid medium, and cultured at 37°C with shaking for 16 hours. 1 mL of culture (OD) was taken. 600 ≈1) Mix with an equal volume of 50% (m / v) glycerol to prepare glycerol bacteria (dual plasmid host bacteria), and store in an ultra-low temperature freezer at –80°C as the standard host strain for subsequent mutagenesis experiments.

[0092] In this dual plasmid system, the target vector provides an aadA-GFP reporter module that can be recognized by gRNA and used for phenotypic screening, while the mutagenesis vector expresses CasPol or its components under tetracycline induction. The two work together to achieve a controllable and detectable targeted mutagenesis function.

[0093] Example 6: Determination of the screening concentration of spectinomycin To establish an effective phenotypic screening system for identifying CasPol-mediated aadA function recovery mutants, this invention systematically optimized the screening concentration of spectinomycin (Spc). Using *E. coli* TG1 host bacteria (a dual-plasmid host bacteria with a glutamine mutation at position 12 of aadA to a stop codon, resulting in loss of function) constructed in Example 5 carrying the dual plasmids pCNACE-tet-CasPol and pBAD33J-aadA_Q12-GFP as the test subject, LB solid medium containing different concentrations of Spc (0, 100 μg / mL, 200 μg / mL, and 400 μg / mL) was prepared. OD 600 Equal amounts of bacterial suspension with a concentration of 0.3 were spread on each plate and incubated at 37°C for 16 hours to observe growth.

[0094] The results showed that: (1) the loss-of-function strain still showed weak growth on LB solid medium containing 0 and 100 μg / mL Spc, indicating insufficient screening pressure; (2) the mutant strain did not grow at all on LB solid medium containing 200 μg / mL and 400 μg / mL Spc, indicating that the background growth of non-functional aadA was effectively inhibited.

[0095] To further verify whether a Spc concentration of 200 μg / mL allows the pBAD33J-aadA-GFP strain to grow normally, host bacteria carrying pBAD33J-aadA-GFP (containing the complete aadA coding sequence) (OD600=0.1) were plated on LB solid medium containing a Spc concentration of 200 μg / mL. The results showed that at this concentration, the wild-type strain could form clear and healthy single colonies.

[0096] In summary, Spc spectinomycin at a concentration of 200 μg / mL effectively inhibits the growth of aadA loss-of-function mutants without hindering the survival of functional aadA strains, demonstrating good screening specificity. Therefore, it was selected as the screening concentration for spectinomycin resistance recovery mutants in subsequent mutagenesis experiments.

[0097] Example 7: Concentration optimization of tetracycline-induced CasPol expression To balance the expression level, mutagenesis efficiency, and host bacterial growth status of the CasPol fusion protein, this invention conducted gradient tests on tetracycline (Tet) induction concentrations. The TG1 strain carrying the dual plasmids pCNACE-tet-CasPol and pBAD33J-aadA_Q12-GFP was inoculated into LB liquid medium, and tetracycline was added at concentrations of 0, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL, respectively. The culture was induced at 37°C with shaking for 13 hours. The following procedures were then performed:

[0098] (1) OD of each group of bacterial solutions was measured using a spectrophotometer. 600 To assess the effects of tetracycline on bacterial growth.

[0099] (2) Take OD 600 Equal volumes of bacterial suspension (0.3 g / mL) were spread onto LB solid medium containing 200 μg / mL spectinomycin. After incubation at 37°C for 16 hours, the number of spectinomycin-resistant colonies was counted as a quantitative indicator of CasPol mutagenicity.

[0100] Experimental results showed that: (1) when the tetracycline concentration was ≤5 ng / mL, bacterial growth was not significantly inhibited, and OD 600 Similar to the uninduced group (0 ng / mL); (2) When the concentration is ≥10 ng / mL, the bacterial OD 600 A significant decrease suggests that high concentrations of tetracycline have toxic or metabolic burdens on the host bacteria; (3) The number of resistant recovered colonies reached its peak at 5 ng / mL; when the concentration increased to 10 ng / mL or above, although CasPol expression may be enhanced, the overall number of recovered colonies decreased due to decreased cell viability.

[0101] Taking into account cell growth status, mutagenesis efficiency, and experimental reproducibility, this invention determined 5 ng / mL tetracycline as the optimal induction concentration for subsequent CasPol-mediated targeted mutagenesis experiments.

[0102] Example 8: Comparison of the functional recovery efficiency of aadA_Q12 by different combinations of mutagenesis modules To verify the necessity of the synergistic effect between the targeting cleavage module (SaCas9n-KKH) and the error-prone synthesis module (mPolA) in the CasPol fusion protein for its mutagenic function, this invention constructed four different dual-plasmid systems and systematically compared their ability to restore spectinomycin resistance in the context of premature termination mutation of aadA_Q12. Specifically, the following four mutagenic plasmids were transformed into E. coli TG1 host bacteria already carrying the target vector pBAD33J-aadA_Q12-GFP, forming the following four groups of dual-plasmid strains:

[0103] Control group (empty vector group): empty vector pCNACE-tet (pCNACE-tet vector, without SaCas9n-KKH or mPolA).

[0104] Contains only the cutting module group: pCNACE-tet-Cas (pCNACE-tet-Cas vector, expressing only SaCas9n-KKH).

[0105] Contains only the mutagenic module group: pCNACE-tet-Pol (pCNACE-tet-Pol vector, expressing only mPolA).

[0106] Complete system group: pCNACE-tet-CasPol (pCNACE-tet-CasPol vector, expressing SaCas9n-KKH–linker–mPolA fusion protein).

[0107] After activation, each group of strains was used with initial OD. 600 =0.3 was inoculated into 10 mL of LB liquid medium, with two parallel treatments per group: (1) Induction group: 5 ng / mL tetracycline (Tet) was added to initiate the expression of the mutagenesis module.

[0108] (2) Non-induction group: no tetracycline was added as a negative control.

[0109] All cultures were incubated at 37°C and 220 rpm with shaking for 13 hours. OD values ​​were then collected. 600 An equal volume of bacterial suspension with a concentration of 0.3 was spread onto LB agar containing 200 μg / mL spectinomycin. After incubation at 37°C for 16 hours, the number of colonies that recovered resistance was counted (see results below). Figure 4 ).

[0110] The experimental results are as follows: (1) No observable resistant colonies were generated in the empty vector group and the group containing only the cutting module (pCNACE-tet and pCNACE-tet-Cas) under induced or non-induced conditions, indicating that simple DNA cutting or non-functional vectors cannot drive the recovery of aadA function.

[0111] (2) Only the mutagenic module group (pCNACE-tet-Pol) showed a small number of resistant colonies under induction conditions, suggesting that free mPolA may achieve very limited functional recovery through inefficient random mutation, but the efficiency is significantly low.

[0112] (3) The complete CasPol systemome (pCNACE-tet-CasPol) produced a large number of spectinomycin-resistant colonies under tetracycline induction conditions, while the non-inducible group showed no significant growth, indicating that the synergistic effect of CasPol's targeted cleavage and local error-prone synthesis can efficiently mediate reversion mutations or functional compensation mutations at the aadA_Q12 site.

[0113] In summary, only CasPol fusion proteins that simultaneously possess the ability to target DNA cleavage sites and synthesize adjacent error-prone DNA can efficiently achieve site-directed mutagenesis and resistance recovery under tetracycline induction. This result fully demonstrates the key advantages of the fusion strategy designed in this invention in improving mutagenesis specificity and efficiency.

[0114] Example 9: Quantitative Calculation of Target Gene Mutation Rate To objectively evaluate the efficiency of the CasPol mutagenesis system, this invention employs the maximum likelihood estimation method (Ma–Sarkar–Stewart, MSS method) in the FALCOR (Fluctuation AnaLysis CalculatOR) online tool to quantitatively analyze the mutation rate at the target site.

[0115] Using pCNACE-tet-CasPol / pBAD33J-aadA_Q12-GFP dual-plasmid host bacteria as the experimental system, the bacteria were induced and cultured in LB liquid medium containing 5 ng / mL tetracycline at 37°C and 220 rpm for 13 hours. After the culture was completed, the OD of the bacterial culture was measured. 600 Different dilutions of bacterial suspension were then spread onto the following two types of plates:

[0116] (1) Total viable cell count plate: LB solid medium containing 50 μg / mL kanamycin + 50 μg / mL chloramphenicol (used to maintain the stability of the two plasmids and count the total number of viable cells).

[0117] (2) Anti-resistance recovery mutant counting plate: In addition to the above double antibiotics, 200 μg / mL spectinomycin (for screening mutants with restored aadA function) was added.

[0118] All plates were incubated at 37°C for 16 hours before colony counting. To ensure statistical reliability, only plates with colony counts ranging from 100 to 200 were used for calculation. The colony count was determined based on the spread volume and the OD of the bacterial culture. 600 The total number of viable bacteria (Nt) and the number of spectinomycin-resistant regenerated bacteria (Nm) per milliliter of culture were then calculated.

[0119] The Nm values ​​obtained from multiple parallel experiments are input into the FALCOR tool (MSS method), and the mutation rate (μ) is calculated in conjunction with the corresponding Nt. For example... Figure 5 As shown, the mutation rate of tetracycline-induced CasPol in the experimental group reached 3.57 × 10⁻⁶. −6 The levels were significantly higher than the background level, indicating that the system can efficiently induce heritable functional recovery mutations at the target site.

[0120] Example 10: Phenotypic verification and mutation site identification of resistance-recovered colonies To confirm whether spectinomycin resistance recovery stemmed from a precise reversion mutation at the Q12 site of the aadA gene, this invention performed dual phenotypic and genotypic verification on the resistant positive clones.

[0121] First, positive colonies grown on LB solid medium containing kanamycin (50 μg / mL), chloramphenicol (50 μg / mL), and spectinomycin (200 μg / mL) were placed under a UV excitation source (395 nm) to observe fluorescence signals. Figure 6 As shown in (B), all test colonies exhibited obvious green fluorescence, indicating normal expression of the GFP reporter gene. This suggests that the mutation in the aadA region did not disrupt the downstream reading frame or transcriptional readthrough, supporting functional recovery.

[0122] Subsequently, several resistant positive clones were randomly selected, and their target vector plasmids were extracted. Sanger sequencing was then performed on the region of the aadA gene containing the Q12 site. Sequencing results ( Figure 7 (B) Figure 7 (C) Figure 7 (D) shows that the originally introduced stop codon TAA (corresponding to Q12) has been reverted to wild-type CAA, meaning the glutamine coding sequence has been reconstructed. This reversion mutation directly restores the complete open reading frame of the aadA protein, thus explaining the recurrence of spectinomycin resistance.

[0123] In summary, the resistance-recovered colonies exhibit both the expected fluorescent phenotype and precise target site reversion mutations, fully demonstrating that the CasPol system can mediate high-fidelity, site-specific functional reversion mutagenesis, rather than random global mutations.

[0124] Example 11: Analysis of the effective range of the CasPol mutagenesis system To systematically evaluate the mutagenicity of the CasPol mutagenesis system at different distances from the target site, this invention utilizes a series of aadA (SEQ ID NO.11 and SEQ ID NO.12) to prematurely terminate mutant target vectors (Q12, R22, R57, R84, E87, Q108, Q160), with mutation sites located at 23bp, 53bp, 158bp, 239bp, 248bp, 311bp, and 467bp downstream of the 3′ end of the gRNA recognition sequence, respectively, to conduct spectinomycin resistance recovery experiments. Specific information is as follows:

[0125] aadA-Q12* (SEQ ID NO.13 and SEQ ID NO.114), aadA-R22* (SEQ ID NO.15 and SEQ ID NO.16), aadA-R57* (SEQ ID NO.17 and SEQ ID NO.18), aadA-R84* (SEQ ID NO.19 and SEQ ID NO.20), adA-E87* (SEQ ID NO.19 and SEQ ID NO.20) NO.21 and SEQ ID NO.22), aadA-Q108* (SEQ ID NO.23 and SEQ ID NO.234) and aadA-Q160* (SEQ ID NO.25 and SEQ ID NO.26).

[0126] All dual-plasmid host bacteria (containing pCNACE-tet-CasPol and the corresponding aadA mutant target vector) were cultured for 13 hours after induction with 5 ng / mL tetracycline, and then plated on selective plates containing 200 μg / mL spectinomycin. Figure 8 As shown, all test sites produced positive colonies with restored resistance, indicating that the CasPol system can induce functional reversion mutations over a wide genome.

[0127] The mutagenicity of each mutation site was further quantitatively calculated using the FALCOR tool (MSS maximum likelihood method) (results are shown in...). Figure 9 Data shows:

[0128] (1) In the region within 300 bp of the gRNA recognition site (23 bp ~ 248 bp), the mutation rate remained at 10. −5 Up to 10 −6 The level is relatively high.

[0129] (2) When the distance is extended to 311bp and 467bp, the mutation rate decreases but is still significantly higher than the background level, indicating that CasPol has the ability to induce distant mutagenesis.

[0130] In contrast, the existing EvolvR system based on enCas9-PolI3M exhibits a rapid decay in mutagenesis rate (typically <10) at sites more than 50 bp away. −7 ), and the effective window of action is extremely limited.

[0131] In summary, the CasPol fusion system constructed in this invention not only maintains high mutagenesis efficiency (102) −5 ~10 −6 Furthermore, it significantly expands the effective mutagenesis window from approximately 50 bp to at least 239 bp, greatly enhancing the spatial coverage of targeted mutagenesis. This characteristic gives it a significant advantage in applications requiring saturation mutations or functional scanning of large gene regions.

[0132] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0133] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

[0134] Among them, SEQ ID NO.1: (gRNA scaffold (DNA)): TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAGCAGCGGCATGAGGGAAGCGTTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGATTTTTTTCCGAAAAAAAAACCCCGCCCTGACAGGGCGGGGTTTTTTTTTAATTAACAGAAAATTATTTTAAATTTCCTCCTCGCTGCGG.

[0135] SEQ ID NO.2 (SaCas9n-KKH (DNA, length: 3159bp)):

[0136] SEQ ID NO.3 (SaCas9n-KKH (protein, length: 1053aa)):

[0137] SEQ ID NO.4 (mPolA (DNA, length: 2784bp)):

[0138] SEQ ID NO.5 (mPolA (protein, length: 928 aa)): MVQIPQNPLILVDGSSYLYRAYHAFPPLTNSAGEPTGAMYGVLNMLRSLIMQYKPTHAAVVFDAKGKTFRDELFEHYKSHRPPMPDDLRAQIEPLHAMVKAMGLPLLAVSGVEADDVIGTLAREAEKAGRPVLISTGDKDMAQLVTPNITLINTMTNTILGPEEVVNKYGVPPELIIDFLALMGDSSDNIPGVPGVGEKTAQALLQGLGGLDTLYAEPEKIAGLSFRGAKTMAAKLEQNKEVAYLSYQLATIKTDVELELTCEQLEVQQPAAEELLGLFKKYEFKRWTADVEAGKWLQAKGAKPAAKPQETSVADEAPEVTATVISYDNYVTILDEETLKAWIAKLEKAPVFAFDTETDSLDNISANLVGLSFAIEPGVAAYIPVAHDYLDAPDQISRERALELLKPLLEDEKALKVGQNLKYARGILANYGIELRGIAFDTMLESYILNSVAGRHDMDSLAERWLKHKTITFEEIAGKGKNQLTFNQIALEEAGRYAAEDADVTLQLHLKMWPDLQKHKGPLNVFENIEMPLVPVLSRIERNGVKIDPKVLHNHSEELTLRLAELEKKAHEIAGEEFNLSSTKQLQTILFEKQGIKPLKKTPGGAPSTSEEVLEELALDYPLPKVILEYRGLAKLKSTYTDKLPLMINPKTGRVHTSYHQAVTATGRLSSTDPNLQNIPVRNEEGRRIRQAFIAPEDYVIVSADYSQNELRIMAHLSRDKGLLTAFAEGKDIHRATAAEVFGLPLETVTSEQRRSAKRINFGLIYGMSAFGLARQLNIPRKEAQKYMDLYFERYPGVLEYMERTRAQAKEQGYVETLDGRRLYLPDIKSSNGARRAAAERAAINAPMQGTAADIIKRAMIAVDAWLQAEQPRVRMIMQVHDELVFEVHKDDVDAVAKQIHQLMENCTRLDVPLLVEVGSGENWDQAH。

[0139] SEQ ID NO.6 (32aa-linker (DNA, length: 96bp)): TCCGGTGGTTCAAGTGGAGGTTCCTCAGGCAGCGAAACGCCGGGCACCAGCGAGAGCGCGACGCCGGAAAGCTCTGGTGGTTCTTCTGGTGGCTCC.

[0140] SEQ ID NO.7 (32aa-linker (protein, length: 32aa)): SGGSSGGSSGSETPGTSESATPESSGGSSGGS.

[0141] SEQ ID NO.8 (CasPol (protein, length: 2012aa)):

[0142] SEQ ID NO.9 (GFP (DNA, length: 735 bp)): TCCGGTGGTTCAAGTGGAGGTTCCCGTAAAGGCGAAGAGCTGTTCACTGGTGTCGTCCCTATTCTGGTGGAACTGGATGGTGATGTCAACGGTCATAAGTTTTCCGTGCGTGGCGAGGGTGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGTTCATCTGTACTACTGGTAAACTGCCGGTTCCTTGGCCGACTCTGGTAACGACGCTGACTTATGGTGTTCAGTGCTTTGCTCGTTATCCGGACCATATGAAGCAGCATGACTTCTTCAAGTCCGCCATGCCGGAAGGCTATGTGCAGGAACGCACGATTTCCTTTAAGGATGACGGCACGTACAAAACGCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTAAACCGCATTGAGCTGAAAGGCATTGACTTTAAAGAGGACGGCAATATCCTGGGCCATAAGCTGGAATACAATTTTAACAGCCACAATGTTTACATCACCGCCGATAAACAAAAAAATGGCATTAAAGCGAATTTTAAAATTCGCCACAACGTGGAGGATGGCAGCGTGCAGCTGGCTGATCACTACCAGCAAAACACTCCAATCGGTGATGGTCCTGTTCTGCTGCCAGACAATCACTATCTGAGCACGCAAAGCGTTCTGTCTAAAGATCCGAACGAGAAACGCGATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGCATGGTATGGATGAACTGTACAAA。

[0143] SEQ ID NO.10 (GFP (protein, length: 245 aa)): SGGSSGGSRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK。

[0144] SEQ ID NO.11 (aadA (DNA, length: 804 bp)): ATGGGCAGCAGCGGCATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGAGGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACGTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAA。

[0145] SEQ ID NO.12 (aadA (protein, length: 268 aa)): MGSSGMREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPHSDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKEVAADWAMERLPAQYQPVIREARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK。

[0146] SEQ ID NO.13 (aadA - Q12* (DNA, length: 804 bp)): ATGGGCAGCAGCGGCATGAGGGAAGCGGTGATCGCCGAAGTATCGACTTAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGAGGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACGTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAA。

[0147] SEQ ID NO.14 (aadA-Q12* (Protein, length: 268 aa)): MGSSGMREAVIAEVST*LSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPHSDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKEVAADWAMERLPAQYQPVIREARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK。

[0148] SEQ ID NO.15 (aadA-R22* (DNA, length: 804 bp)): ATGGGCAGCAGCGGCATGAGGGAAGCGGTGATCGCCGAGGTCTCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGTGACATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAA。

[0149] SEQ ID NO.16 (aadA-R22* (Protein, length: 268 aa)): MGSSGMREAVIAEVSTQLSEVVGVIE*HLEPTLLAVHLYGSAVDGGLKPHSDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQPVILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK。

[0150] SEQ ID NO.17 (aadA-R57* (DNA, length: 804 bp)): ATGGGCAGCAGCGGCATGAGGGAAGCGGTGATCGCCGAGGTCTCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACGGTGTGACTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAA。

[0151] SEQ ID NO.18 (aadA-R57* (Protein, length: 268 aa)): MGSSGMREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPHSDIDLLVTVTV*LDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQPVILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK。

[0152] SEQ ID NO.19 (aadA-R84* (DNA, length: 804 bp)): ATGGGCAGCAGCGGCATGAGGGAAGCGGTGATCGCCGAGGTCTCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCTGAGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAA。

[0153] SEQ ID NO.20 (aadA-R84* (Protein, length: 268 aa)): MGSSGMREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPHSDIDLLVTVTVRLDETTRRALINDLLETSASPGESEIL*AVEVTIVVHDDIIPWRYPAKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQPVILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK。

[0154] SEQ ID NO.21 (aadA-E87* (DNA, length: 804 bp)): ATGGGCAGCAGCGGCATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTATAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGAGGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACGTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAA。

[0155] SEQ ID NO.22 (aadA-E87* (protein, length: 268 aa)): MGSSGMREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPHSDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAV*VTIVVHDDIIPWRYPAKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKEVAADWAMERLPAQYQPVIREARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK。

[0156] SEQ ID NO.23 (aadA - Q108* (DNA, length: 804 bp)): ATGGGCAGCAGCGGCATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGTAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGAGGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACGTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAA。

[0157] SEQ ID NO.24 (aadA - Q108* (Protein, length: 268 aa)): MGSSGMREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPHSDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAKREL*FGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKEVAADWAMERLPAQYQPVIREARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK。

[0158] SEQ ID NO.25 (aadA-Q160* (DNA, length: 804 bp)): ATGGGCAGCAGCGGCATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAATAAGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGAGGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACGTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAA。

[0159] SEQ ID NO.26 (aadA - Q160* (protein, length: 268 aa)): MGSSGMREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPHSDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPE*DLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKEVAADWAMERLPAQYQPVIREARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK。

[0160] SaCas9-KKH PAM (DNA, length: 6bp): NNGRRT。

[0161] SEQ ID NO.27 (SaCas9 gRNA (DNA, length: 20bp)): AGCAGCGGCATGAGGGAAGC。

[0162] SEQ ID NO.28 (J23101 promoter (DNA, length: 35bp)): TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGC。

Claims

1. A SaCas9n-mPolA fusion protein, characterized in that, The SaCas9n-mPolA fusion protein is composed of a SaCas9n domain, a flexible linker peptide, and an mPolA domain sequentially linked from the N-terminus to the C-terminus. The amino acid sequence of the SaCas9n domain is shown in SEQ ID NO.3; The amino acid sequence of the flexible linker peptide is shown in SEQ ID NO.7; The amino acid sequence of the mPolA domain is shown in SEQ ID NO.

5.

2. A mutagenic vector, characterized in that, It contains a nucleotide sequence encoding the SaCas9n-mPolA fusion protein of claim 1.

3. A directed in vivo evolution system for a designated region of genes, characterized in that, It includes the mutagenesis vector and target vector as described in claim 2, wherein the mutagenesis vector and target vector are two mutually compatible plasmids that can coexist stably in the same host cell; The target vector comprises a pBAD promoter, a target sequence, an aadA resistance marker gene, and a GFP reporter gene; the target sequence comprises a PAM sequence recognized by the SaCas9n domain, and the nucleotide sequence of the PAM sequence is shown in SEQ ID NO.

27.

4. A nucleotide sequence encoding the SaCas9n-mPolA fusion protein of claim 1.

5. A dual-plasmid host bacterium, characterized in that, It includes the mutagenic vector and target vector as described in claim 3.

6. A method for directed in vivo evolution of genes in a designated region, characterized in that, Includes the following steps: The mutagenic vector and target vector described in claim 5 were co-transformed into the host bacteria to construct the dual-plasmid host bacteria, and positive clones were obtained by double screening with chloramphenicol and kanamycin. Adding tetracycline induces the expression of the SaCas9n-mPolA fusion protein. The SaCas9n-mPolA fusion protein targets and binds to the target sequence under the guidance of guide RNA. The SaCas9n domain performs single-strand cleavage of the non-target strand, and the mPolA domain performs error-prone DNA synthesis at the cleavage site, introducing mutations. Through resistance screening, evolutionary mutants with restored or enhanced function of the aadA resistance marker gene were obtained.

7. The method according to claim 6, characterized in that, The conditions for inducing the expression of the SaCas9n-mPolA fusion protein by adding tetracycline are as follows: The concentration of tetracycline was 4.5 ng / mL to 5.5 ng / mL, and the induction time was 4 to 16 hours.

8. The use of the fusion protein of claim 1, the gene-directed evolution system of the designated region of claim 3, the nucleotide sequence of claim 4, the mutagenesis vector of claim 2, or the dual-plasmid host bacterium of claim 5 in the preparation of a product that has been modified by directed evolution or the gene encoding the protein.

9. The application according to claim 8, characterized in that, The products include enzyme preparations, biocatalysts, diagnostic reagents, and therapeutic proteins.

Images