Cytosine base editing system and method applied to mycobacteria

Abstract

The invention discloses a cytosine base editing system and method applied to mycobacteria, and belongs to the technical field of genetic engineering. A CRISPR-Cas12a (clustered regularly interspaced short palindromic repeats-CRISPR-Cas12a) technology is coupled with a cytosine nucleoside deaminase system, so that efficient genetic manipulation on a mycobacterium gene is realized. According to the invention, FnCas12a derived from Francisella is subjected to multi-sequence alignment and structural rational analysis for successful design and modification to obtain an FnCas12a mutant dFnCas12a, cytosine nucleoside deaminase from different sources is screened, an optimal single plasmid base editing system pBh3A is constructed, and cytosine base editing of a target sequence is realized. Meanwhile, the influence of the quantity of uracil DNA glycosylase inhibitors (UGI) and the types of promoters on the base editing efficiency is researched, and an effective technical means is provided for researching mycobacterium genome editing.

Description

Technical Field

[0001] The present invention relates to a gene editing system, specifically a cytosine base editing system and method applied to mycobacteria, and more particularly to a system and method for performing cytosine base editing on mycobacteria using cytosine nucleoside deamination technology based on the CRISPR / Cas12a system. Background Art

[0002] Steroidal drugs are a class of compounds with a basic skeleton of cyclopentanepolyhydrophenanthrene and have extremely important clinical applications. This class of drugs mainly includes glucocorticoids, mineralocorticoids, sex hormones, and vitamin D derivatives, and their effects cover almost all important systems of the human body. This makes steroidal drugs the second largest class of drugs after antibiotics. Currently, steroidal drugs and steroidal drug intermediates have a large production and export scale in my country. Research on steroid synthesis, extraction, and excellent bacterial strains has never been interrupted. However, compared with advanced countries in the world, there are still many shortcomings, resulting in a persistent low-end position in this field. Therefore, there is an urgent need to break the foreign technology monopoly in the production and research and development of steroidal drugs.

[0003] The synthesis of steroidal drugs relies primarily on a series of key steroidal intermediates, all of which share the basic sterane nucleus structure. The most important intermediates include 4-androstene-3,17-dione (AD), 1,4-androstadiene-3,17-dione (ADD), and 9-hydroxy-4-androstene-3,17-dione (9-OH-AD). Other intermediates include 22-hydroxy-23,24-dinorcholene-3-one (4-HBC), 22-hydroxy-23,24-dinorcholene-1,4-diene-3-one (1,4-HBC), and 9,22-dihydroxy-23,24-dinorcholene-3-one (9-OH-4-HBC), each bearing specific modified groups. These intermediates can be further synthesized through various chemical or biological transformation pathways to yield a variety of pharmaceutically valuable steroidal compounds.

[0004] Mycobacterium neoaurum is a key microbial foundation for the industrial production of steroid drugs. Its endogenous metabolic pathways play an irreplaceable role in the biosynthesis of steroid compounds. However, this strain faces significant genetic engineering bottlenecks: first, its inherent cellular barrier system hinders the stable integration and efficient expression of exogenous genes; second, existing gene editing tools have significant limitations. Current mainstream allele replacement techniques face two challenges: first, low homologous recombination efficiency due to low endogenous recombinase activity; second, the method requires the introduction of a resistance selection marker, limiting the feasibility of multiple rounds of gene editing. While the pNIL / pGOAL suicide vector system, developed based on homologous recombination principles, addresses the issue of marker carryover, editing efficiency remains insufficient due to the host's limited DNA repair capacity. These technical bottlenecks severely hinder progress in optimizing the production performance of Mycobacterium neoaurum through metabolic engineering. Therefore, the development of new, efficient, and precise gene editing systems has become a key scientific challenge that urgently needs to be overcome in this field.

[0005] In recent years, significant progress has been made in mycobacterial gene editing technology, especially the introduction of the CRISPR-Cas system, which has brought revolutionary breakthroughs in this field. Studies have shown that the gene editing system based on the synergistic action of homologous recombination (HR) and CRISPR-Cas12a can efficiently achieve various genetic modifications such as point mutations, gene deletions, and insertions in Mycobacterium smegmatis. In addition, scientists have also developed editing tools based on the CRISPR-Cas-mediated non-homologous end joining (NHEJ) mechanism, which can achieve markerless gene deletion in mycobacteria. However, these technologies still have obvious limitations: the existing CRISPR-Cas system cannot achieve precise point mutation introduction in mycobacteria, and it is difficult to complete fine genetic manipulation, which to some extent restricts the in-depth development of mycobacterial functional genomic research.

[0006] The emergence of base editing technology has revolutionized the way genome editing is accomplished. Unlike traditional homologous recombination (HR) and non-homologous end joining (NHEJ) techniques, which rely on double-strand breaks (DSBs) or donor DNA templates, base editors achieve precise editing by cleverly fusing an inactivated Cas nuclease with a single-stranded DNA deaminase. Currently, there are two main types of base editors: cytosine base editors (CBEs) fused to cytosine deaminases (such as the APOBEC family), and adenine base editors (ABEs) fused to adenine deaminase (TadA). In CBEs, the APOBEC deaminase is fused to the catalytically impaired Cas12a protein. When targeted to a specific genomic locus under the guidance of crRNA, the system forms a unique "R-loop" structure, exposing a single-stranded region of the target DNA. APOBEC deaminates cytosine (C) within this single-stranded window, converting it to uracil (U), which is then recognized as thymine (T) during DNA replication. However, in bacterial cells, this U:G mismatch is typically cleared by uracil N-glycosylase (UNG) through the base excision repair (BER) pathway. To protect the edited product, the researchers fused a uracil DNA glycosylase inhibitor (UGI) to the C-terminus of Cas12a, effectively inhibiting UNG activity and prompting cells to convert U:G to U:A via the mismatch repair pathway, ultimately achieving a stable C:G to T:A conversion during DNA replication. This will be an accessible, simple, and efficient genetic tool that will further promote the modification and research of steroid-producing Mycobacterium chromogenes. Summary of the Invention

[0007] Purpose of the invention: The purpose of the present invention is to design and provide a technical solution for a cytosine base editing system and method for use in Mycobacterium chromosomum. This invention constructs a fast, efficient and stable pBh3A base editing system in Mycobacterium sp. LY-1 CGMCC No. 13031 for the first time. The system is based on mutated dFnCas12a (L914A, D917A and E1006A) and uses pJV53 as a plasmid template to connect hAPOBEC3A-dFnCas12a-UGI and crRNA array. The base editing efficiency of the CRISPR-Cas12a system is then improved by optimizing the promoter and UGI quantity.

[0008] The first object of the present invention is to provide an FnCas12a mutant, the protein sequence of the FnCas12a mutant is shown in SEQ ID No: 3, and is recorded as dFnCas12a.

[0009] The nucleotide sequence encoding the FnCas12a mutant of the present invention is shown in SEQ ID No: 4.

[0010] Alternatively, in one embodiment of the present invention, the FnCas12a mutant is obtained by subjecting the FnCas12a having an amino acid sequence derived from Francisella U112 as shown in SEQ ID No: 2 to the following mutations: (a) Leucine at position 914 was mutated to alanine; (b) Aspartic acid at position 917 was mutated to alanine; (c) Glutamic acid at position 1006 was mutated to alanine.

[0011] Optionally, in one embodiment of the present invention, the nucleotide sequence of FnCas12a is shown in SEQ ID No: 1.

[0012] A second object of the present invention is to provide a fusion protein, which is a sequence formed by fusion of cytosine deaminase hAPOBEC3A, dFnCas12a and uracil glycosidase inhibitor UGI, wherein the dFnCas12a is a FnCas12a mutant as described above.

[0013] Optionally, in one embodiment of the present invention, the gene sequence encoding cytidine deaminase hAPOBEC3A is shown as SEQ ID No: 5.

[0014] Optionally, in one embodiment of the present invention, the gene sequence encoding the uracil DNA glycosylase inhibitor UGI is shown in SEQ ID No: 6; Optionally, in one embodiment of the present invention, the cytosine deaminase hAPOBEC3A is connected to the N-terminus of dFnCas12a through a linker XTEN encoding 16 amino acids (sequence: Ser-Gly-Ser-Glu-Thr-Pro-Gly-Thr-Ser-Glu-Ser-Ala-Thr-Pro-Glu-Ser) as shown in SEQ ID No: 7.

[0015] Optionally, in one embodiment of the present invention, the uracil glycosidase inhibitor UGI is connected to the C-terminus of dFnCas12a via a linker SGGS encoding 4 amino acids (Ser-Gly-Gly-Ser) as shown in SEQ ID No: 8.

[0016] Optionally, in one embodiment of the present invention, the cytidine deaminase hAPOBEC3A, dFnCas12a and UGI are promoted by a strong promoter hsp60, and the nucleotide sequence of the strong promoter hsp60 is shown in SEQ ID No:9.

[0017] The third object of the present invention is to provide a base editing system for mycobacteria, which is a CRISPR-dFnCas12a-assisted single-plasmid cytosine base editing system pBh3A, which includes any of the fusion proteins described above and its corresponding crRNA array.

[0018] Optionally, in one embodiment of the present invention, the cytosine base editing plasmid is a replicative shuttle vector. In one embodiment of the present invention, the replicative shuttle vector contains the Mycobacterium replication origin pAL5000 and the Escherichia coli replication origin ori.

[0019] Optionally, in one embodiment of the present invention, the crRNA array encoded on the cytosine base editing system is composed of a direct repeat sequence-target sequence-direct repeat sequence unit sequence, and its nucleotide sequence is shown in SEQ ID No: 10.

[0020] Optionally, in one embodiment of the present invention, the crRNA array encoded on the cytosine base editing system is initiated by the mycobacterium endogenous promoter pmn1, and its sequence is shown in SEQ ID No:11.

[0021] A fourth object of the present invention is to provide a method for single-plasmid cytosine base editing in Mycobacteria based on the above-mentioned cytosine base editing system, comprising the following steps: 1) Preparing mycobacteria into competent cells capable of electroporation; 2) ligating the target gene targeting sequence to the crRNA interaction element on the cytosine base editing plasmid to obtain a cytosine base editing plasmid containing a complete crRNA sequence; 3) The cytosine base editing plasmid containing the crRNA sequence prepared in step 2) was transformed into the competent cells prepared in step 1), and after kanamycin resistance screening, the cells were inoculated in LB medium for culture. The transformants edited for the target gene fragment were obtained by double verification by first-generation Sanger sequencing and Illumina second-generation sequencing.

[0022] A fifth object of the present invention is to provide a dFnCas12a mutant as described above, or a fusion protein as described in any one of the above, or an application of a base editing system as described in any one of the above in gene editing, mutant construction, biosynthesis or metabolic regulation.

[0023] Technical Effect: This invention utilizes dFnCas12a (L914A, D917A, and E1006A) in combination with the cytidine deaminase hAPOBEC3A to form the base editing system pBh3A, enabling efficient genetic manipulation of mycobacterial genes. Furthermore, the cytosine base editing system of this invention improves the base editing efficiency of the CRISPR-Cas12a system by optimizing the number of promoters and UGIs, providing an effective technical means for studying Mycobacterium tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the vector structure of the single-plasmid cytosine base editing system constructed in the present invention.

[0025] Figure 2 Design and modification of dFnCas12a in Example 1 of the present invention.

[0026] Figure 3 Screening of cytosine deaminase in Example 2 of the present invention.

[0027] Figure 4 Effect of promoter optimization on the CRISPR-dFnCas12a cytosine base editing system in Example 3 of the present invention.

[0028] Figure 5 Effect of UGI quantity optimization on the CRISPR-dFnCas12a cytosine base editing system in Example 4 of the present invention. DETAILED DESCRIPTION

[0029] The specific construction process of the cytosine base editing system of the present invention is described in detail below with reference to the accompanying drawings through examples, and the usage method and experimental results of the cytosine base editing system of the present invention are further explained through examples.

[0030] The pJV53 vector involved in the following examples was purchased from Hunan Fenghui Biotechnology Co., Ltd.; the one-step cloning reagent ClonExpress II One Step Cloning Kit involved in the following examples was purchased from Nanjing Novozymes Biotechnology Co., Ltd.; the mycobacterium involved in the following examples was Mycobacterium LY-1 CGMCC No. 13031.

[0031] The preparation method of Mycobacterium LY-1 competent cells is as follows: Inoculate the frozen tube with Mycobacterium LY-1 into 10 mL of LB liquid medium and incubate at 30°C with shaking for 64 h to obtain a seed solution. Transfer the seed solution to 50 mL of competent medium at a 2% inoculum volume and incubate at 30°C with shaking for 4-6 h to obtain a bacterial suspension. Pre-chill the bacterial suspension, 10% glycerol, a dry, sterile electroporation cuvette, and a sterilized 50 mL centrifuge tube on ice for 20-30 min. Transfer the pre-chilled bacterial suspension into a centrifuge tube in a clean bench and centrifuge at 5000 rpm and 4°C for 10 min. Discard the supernatant, add 30 mL of 10% glycerol, resuspend the cells, and pre-chill on ice for 15 min. Repeat this step twice, discard the supernatant, add 2 mL of 10% glycerol, resuspend the cells, and aliquot into EP tubes, 100 µL per tube, to obtain Mycobacterium LY-1 CGMCC No. 13031 competent cells.

[0032] The transformation method for Mycobacterium LY-1 is as follows: In a clean bench, add the plasmid to be transformed into Mycobacterium LY-1 competent cells, mix well, and place on ice for 30 minutes. After 30 minutes, transfer all the competent cells to a pre-cooled electroporation cup, quickly wipe the outer wall of the cup dry, place it in an electroporator, and electroporate twice at 2.5 kV for 5 ms. After the electroporation, add 1 mL of LB medium to the electroporation cup and mix well. Transfer all the cells to an EP tube and resuscitate on a shaker at 30°C for 2-3 hours. After resuscitation, collect the cells by centrifugation, remove part of the supernatant, spread on a slant / solid culture medium containing resistance, and culture in an incubator upside down at 30°C for 5-7 days to obtain transformed Mycobacterium LY-1 CGMCC No. 13031.

[0033] Base editing is primarily determined through first-generation Sanger sequencing and second-generation Illumina sequencing. Sanger sequencing, based on the chain termination method, utilizes ddNTPs (dideoxynucleotides) to randomly terminate DNA chain extension, generating fragments of varying lengths. It is primarily produced by Tianlin Biotechnology Co., Ltd. Second-generation Illumina sequencing, based on sequencing by synthesis (SBS), involves bridge amplification of DNA fragments into clusters, incorporation of fluorescently labeled dNTPs one by one, and base determination through imaging. It is primarily produced by Suzhou Genewise Biotechnology Co., Ltd. Database construction using the Illumina second-generation sequencing platform requires analysis of approximately 20,000,000–30,000,000 reads per sample.

[0034] The detection method of base editing efficiency needs to be determined by second-generation Illumina sequencing: The calculation formula for base editing efficiency is as follows: Base editing efficiency = (number of samples in which C mutated to T / total number of samples) × 100%.

[0035] Example 1: Design and modification of dFnCas12a This example is based on the CRISPR-Cas12a genome editing system composed of FnCas12a from Francisella U112 and crRNA, which was previously excavated in the laboratory. The optimal mutation amino acid site is determined by multiple sequence alignment and Cas12a-dependent structural analysis, eliminating the cleavage function of Cas12a in mycobacteria, realizing the construction of dFnCas12a, and providing a protein basis for Example 2.

[0036] The laboratory constructed the CRISPR / Cas12a genome editing system in which Cas12a was derived from the FnCas12a gene of Francisella U112, whose amino acid sequence is shown in SEQ ID No: 2 and the encoding nucleotide sequence is shown in SEQ ID No: 1.

[0037] There are two reasons why CRISPR / Cas12a cannot knock out the target gene: first, Cas12a loses its ability to cut DNA but retains its ability to bind to crRNA; second, mutations hinder the binding of Cas12a to crRNA, preventing it from forming the Cas12a / crRNA complex and correctly positioning and functioning. Therefore, when screening for mutant amino acid sites, it is necessary to avoid the crRNA-Cas12a binding region.

[0038] On the one hand, the embodiment of the present invention performed a multiple sequence alignment of Cas12a from different sources, including Lachnospiraceae bacterium, Acidaminococcus sp., Scytonema hofmanni, Flavobacteriales bacterium, and Francisella novicida U112, and identified 5 potential mutation sites of amino acids D870, H881, L914, E1006, and Y1075, such as Figure 2 As shown in A.

[0039] On the other hand, the embodiments of the present invention are based on the structural analysis of FnCas12a. Figure 2 As shown in Figure 2, the cleavage activity regions of FnCas12a were concentrated in RuvC-Ⅰ (892-953), RuvC-Ⅱ (971-1078), and RuvC-Ⅲ (1254-1300), resulting in three potential mutation sites, including L914, E1006, and Y1075.

[0040] In order to quickly examine whether the three amino acid sites play a key role in the cutting function of FnCas12a, the CRISPR / Cas12a genome editing plasmid pEcrRNA was used as a template to perform step-by-step alanine mutations on FnCas12a. The deletion efficiency of the target genes aftA and hsd4a was examined by population verification to reflect whether FnCas12a was gradually inactivated. Regarding the mutation method, first of all, considering that mutations usually mutate from low amino acid sites to high amino acid sites, and secondly, removing the influence of complex side chains, some amino acids (such as lysine and glutamate) carry charges or large side chains, which may affect the local conformation after mutation. Alanine mutation only removes the side chain function and retains the main chain structure. Therefore, this study chose the alanine mutation method.

[0041] As endogenous genes of Mycobacterium LY-1, knockout of hsd4A (17β-hydroxysteroid dehydrogenase gene) and aftA (membrane protein glycosyltransferase gene) does not affect the normal growth of the strain, so these two genes are used as target genes for mutation verification.

[0042] like Figure 2 As shown in C, with the gradual mutation of the three amino acid sites, the knockout efficiency of aftA and hsd4a in this system was completely reduced to 0%.

[0043] The single plasmid editing system pEcrhsd4a was transformed into Mycobacterium LY-1 according to the electroporation method of Mycobacterium, and primers hsd4a-F and hsd4a-R were designed to verify the knockout of the target gene hsd4a. Figure 2 As shown in C, with the gradual mutation of the three amino acid sites, the knockout efficiency of hsd4a in this system was completely reduced to 0%.

[0044] hsd4a-F: 5'-GCACCAACATGCTCGACACCGG-3' (SEQ ID No: 12) hsd4a-R: 5'-CGGTACATCGTCCTCACCCGGG-3' (SEQ ID No: 13) The single plasmid editing system pEcraftA was transformed into Mycobacterium LY-1 according to the electroporation method of Mycobacterium, and primers aftA-F and aftA-R were designed to verify the knockout of the target gene aftA. Figure 2 As shown in C, with the gradual mutation of the three amino acid sites, the knockout efficiency of aftA in this system was completely reduced to 0%.

[0045] aftA-F: 5'-GACAGGTCGGTGAAGACGCCGAC-3' (SEQ ID No: 14) aftA-R: 5'-CGAAGTCGGTGCAGTACTTGGACTTCGAC-3' (SEQ ID No: 15) The above knockout validation experiments of the target genes hsd4a and aftA completely eliminated the knockout function of the CRISPR-Cas12a genome editing system composed of FnCas12a from Francisella U112 and crRNA. Because the screening sites are only in the FnCas12a cleavage active regions RuvC-Ⅰ (892-953), RuvC-Ⅱ (971-1078), and RuvC-Ⅲ (1254-1300), the cleavage function of Cas12a in mycobacteria is eliminated, while the ability of the CRISPR-Cas12a system to bind to the target gene is retained. The amino acid sequence of the mutated FnCas12a is shown in SEQ ID No: 3, and the nucleotide sequence is shown in SEQ ID No: 4.

[0046] Example 2: Screening of cytosine deaminase The purpose of this example is to construct a cytosine base editing system in Mycobacterium LY-1 by screening 5 cytosine deaminases from different sources. Considering that the aftA gene is more conducive to craftA design and convenient experimental operation, this example uses the gene aftA as the target to determine the cytosine base editing range and editing efficiency of CRISPR-dFnCas12a.

[0047] To construct a cytosine base editing system in Mycobacterium LY-1, four cytosine deaminases from the APOBEC and AID families were screened, including hAPOBEC3A (SEQ ID No: 5), rAPOBEC1 (SEQ ID No: 16), hAID (SEQ ID No: 17), and mAID (SEQ ID No: 18). In addition, PmCDA1 (SEQ ID No: 19), which has a wider editing window, was selected.

[0048] In order to determine the base editing ability of the five cytosine deaminases we screened, the cytosine deaminases were respectively connected to the mutant dFnCas12a obtained in the example to form an expression unit, and a linker with a length of 16 amino acids was used to connect them in series (Ser-Gly-Ser-Glu-Thr-Pro-Gly-Thr-Ser-Glu-Ser-Ala-Thr-Pro-Glu-Ser) (the encoding nucleotide is shown in SEQ ID No: 7). Five cytosine base editing systems pBh3A, pBrA, pBhAID, pBmAID and pBPm were successfully constructed, as shown in FIG. Figure 1 As shown in A.

[0049] Using the endogenous gene aftA of Mycobacterium as the target, firstly, first-generation Sanger sequencing was used to determine whether the target sequence craftA (SEQ ID No: 20) showed multiple peaks, such as Figure 3 As shown in A, the cytosine base editing systems identified as mutated are pBh3A, pBrA, and pBmAID.

[0050] On the basis of first-generation sequencing, second-generation high-throughput Illumina sequencing was used to confirm that C12 and C20 of the cytosine base editing systems pBh3A, pBrA, and pBmAID underwent base editing from C to T. Among them, pBh3A had the highest cytosine base editing efficiency, with a base editing efficiency of 54% for C12 and 48.6% for C20. Figure 3 As shown in B, therefore, the subsequent examples are all carried out based on the pBh3A cytosine base editing system.

[0051] Example 3: Effect of Promoter on Cytosine Base Editing System The purpose of this example is to enhance the base editing efficiency of the pBh3A cytosine base editing system. Using plasmid pJV53 as a template, based on the original cytosine base editing system pBh3A, the endogenous promoter pmn2 (SEQ ID No: 21) of the expression unit composed of the deaminase hAPOBEC3A and dFnCas12a was replaced with the constitutive strong promoter hsp60 (SEQ ID No: 11). Primers hsp60-F and hsp60-R were designed to amplify hsp60, and the promoter pmn2 was replaced with hsp60 using a one-step cloning method. Figure 1 B and Figure 4 As shown in A.

[0052] Primer pairs for hsp60 constitutive strong promoter hsp60-F: 5'-ACGCAGACCGGGTGACCACAACGACGCGCC-3' (SEQ ID No: 22) hsp60-R: 5'-TCTCCATCATACTCGCTGCGACGACGGGC-3' (SEQ ID No: 23) The endogenous gene aftA of mycobacteria was used as the target and sequenced directly using the second generation high throughput Illumina. Figure 4 As shown in Figure 3, C12, C16, and C20 of the cytosine base editing system pBh3A all underwent C-to-T base editing, with the base editing efficiency of C12, C16, and C20 being 75.8%, 69.4%, and 78.6%.

[0053] Example 4: Effect of UGI Number on Cytosine Base Editing System The purpose of this example is to enhance the base editing efficiency of the pBh3A cytosine base editing system. Based on the original cytosine base editing system pBh3A, UGI (SEQ ID No: 6) was linked to the C-terminus of dFnCas12a through a linker of 4 amino acids (Ser-Gly-Gly-Ser) (the encoding nucleotide sequence is shown in SEQ ID No: 8), and the effect of the number of UGIs on the cytosine base editing system pBh3A was explored. Primers UGI-F and UGI-R were designed to amplify UGI, and 1 to 3 different numbers of UGIs were linked to the C-terminus of dFnCas12a using a one-step cloning method. Figure 1 C and Figure 5 As shown in A.

[0054] Uracil DNA glycosylase inhibitor (UGI) amplification primer pair UGI-F: 5'-TCTGGTGGTTCTACTAATCTCAGCGAT-3' (SEQ ID No: 24) UGI-R: 5'-GGAACTAGTTTAGAGCATCTTGATTTTGTTCTCCCCGC-3' (SEQ ID No: 25) The endogenous gene aftA of mycobacteria was used as the target and sequenced directly using the second generation high throughput Illumina. Figure 5 As shown in Figure B, when the number of UGIs is 2, C12, C16, and C20 of the cytosine base editing system pBh3A all undergo base editing from C to T, with the base editing efficiency of C12 being 100%, the base editing efficiency of C16 being 100%, and the base editing efficiency of C20 being 90.4%. Continuing to increase the number of UGIs will not increase the cytosine base editing efficiency.

[0055] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A FnCas12a mutant, characterized in that The amino acid sequence of the FnCas12a mutant is shown in SEQ ID No: 3, and is denoted as dFnCas12a.

2. The FnCas12a mutant according to claim 1, wherein The FnCas12a mutant is obtained by subjecting the FnCas12a derived from Francisella U112 with an amino acid sequence as shown in SEQ ID No: 2 to the following mutations: (a) Leucine at position 914 was mutated to alanine; (b) Aspartic acid at position 917 was mutated to alanine; (c) Glutamic acid at position 1006 was mutated to alanine.

3. A fusion protein, characterized in that A sequence formed by fusion of cytosine deaminase hAPOBEC3A, dFnCas12a and uracil glycosidase inhibitor UGI, wherein the dFnCas12a is a FnCas12a mutant as described in claim 1 or 2.

4. The fusion protein according to claim 3, characterized in that The gene sequence encoding cytidine deaminase hAPOBEC3A is shown in SEQ ID No: 5; The gene sequence encoding the uracil DNA glycosylase inhibitor UGI is shown in SEQ ID No: 6; The cytosine deaminase hAPOBEC3A is connected to the N-terminus of dFnCas12a via a linker XTEN encoding 16 amino acids, whose nucleotide sequence is shown in SEQ ID No: 7; The uracil glycosidase inhibitor UGI is connected to the C-terminus of dFnCas12a via a linker SGGS encoding four amino acids as shown in SEQ ID No: 8; The cytosine deaminase hAPOBEC3A, dFnCas12a and UGI are driven by a strong promoter hsp60, and the nucleotide sequence of the strong promoter hsp60 is shown in SEQ ID No:

9.

5. A base editing system for mycobacteria, characterized in that: The invention relates to a CRISPR-dFnCas12a-assisted single-plasmid cytosine base editing system pBh3A, which comprises a fusion protein as described in any one of claims 3 to 4 and a corresponding crRNA array thereof.

6. The base editing system according to claim 5, wherein The single-plasmid cytosine base editing system is derived from the backbone plasmid pJV53, which is a replicative shuttle plasmid containing the Mycobacterium replication origin pAL5000 and the Escherichia coli replicon ori.

7. The base editing system according to claim 5, wherein: The crRNA array encoded on the single plasmid cytosine base editing system is composed of a direct repeat sequence-target sequence-direct repeat sequence unit sequence, and its nucleotide sequence is shown in SEQ ID No:

10.

8. The base editing system according to claim 7, wherein The crRNA array encoded on the single plasmid cytosine base editing system is initiated by the mycobacterium endogenous promoter pmn1, and its nucleotide sequence is shown in SEQ ID No:

11.

9. A method for single-plasmid cytosine base editing in Mycobacteria, comprising performing the following operations using the single-plasmid cytosine base editing system of any one of claims 5 to 8: 1) Preparing mycobacteria into competent cells capable of electroporation; 2) ligating the target gene targeting sequence to the crRNA interaction element on the cytosine base editing plasmid to obtain a cytosine base editing plasmid containing a complete crRNA sequence; 3) The cytosine base editing plasmid containing the crRNA sequence prepared in step 2) was transformed into the competent cells prepared in step 1), and after kanamycin resistance screening, the cells were inoculated in LB medium for culture. The transformants edited for the target gene fragment were obtained by double verification by first-generation Sanger sequencing and Illumina second-generation sequencing.

10. The dFnCas12a mutant according to claim 1, or the fusion protein according to any one of claims 3-4, or the base editing system according to any one of claims 5-8 for use in gene editing, mutant construction, biosynthesis or metabolic regulation.

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