An UNG mutant, fusion protein, base editing system and application thereof that can be used for base editing in Bacillus methanolicus

Abstract

The present invention belongs to the technical field of base editing, and specifically relates to a UNG mutant and a fusion protein, a base editing system and an application thereof that can be used for base editing of Methanolibacillus. The amino acid sequence of the UNG mutant provided by the present invention is shown in SEQ ID NO.1. This UNG mutant can recognize base C in Methanolibacillus and excise it to generate an AP site, thereby realizing base editing of base C. The fusion protein provided by the present invention is composed of the above UNG mutant and single-strand cleavage-inactivated nThermoCas9, and can also edit base C in Methanolibacillus into base T at high temperature. The base editing system provided by the present invention expresses the above fusion protein and gRNA. The base editing technology based on this base editing system provides an important enabling technology for the genetic transformation of Methanolibacillus, and has breakthrough significance for the base editing of thermophilic Methanolibacillus.

Description

Technical Field

[0001] The present invention belongs to the technical field of base editing, and specifically relates to a UNG mutant and a fusion protein, a base editing system and an application thereof that can be used for base editing of Bacillus methanolicus. Background Art

[0002] For a long time, precise editing of genes has relied on homologous recombination (HDR) to complete, but the efficiency of HDR is relatively low in many cells. At the same time, most HDRs need to rely on Cas9 to generate DNA double-strand breaks to improve efficiency, and the inevitable non-homologous end joining (NHEJ) will lead to random mutations. These two disadvantages of HDR have severely restricted the development of the field of gene precise editing. Moreover, both HDR and NEHJ are endogenous DNA repair mechanisms in cells and are not convenient for rational modification.

[0003] The base editing technology emerged in 2016. This technology relies on the precise DNA positioning ability of the CRISPR / Cas9 system and the efficient catalytic ability of base deaminases for precise editing. Compared with the HDR-based gene editing technology, the base editing technology does not rely on the low-efficiency DNA repair process such as HDR, and does not use Cas9 to cause DNA double-strand breaks, and will not trigger random mutations caused by NHEJ. Therefore, it is usually easier to achieve and has higher efficiency.

[0004] The currently disclosed technologies include CBE, ABE and GBE technologies. The CBE and ABE technologies rely on the DNA positioning ability of the CRISPR / Cas9 system. By fusing the Cas9 protein with inactivated double-stranded DNA cleavage function and cytosine (C) or adenine (A) deaminase, the cytosine deaminase or adenine deaminase is positioned at a specific position in the genome, and the C or A at the specific position is catalyzed to undergo a deamination reaction, which is converted into uracil (U) or hypoxanthine (I), and then is recognized as thymine (T) or guanine (G) during DNA replication, respectively realizing the editing of C-T bases and A-G bases. The GBE technology was disclosed in 2020. On the basis of the CBE base editor, uracil glycosylase (UNG) is fused. After C is deaminated to generate U, UNG removes U at this site to generate an apurinic / apyrimidinic (AP) site, and through DNA repair, the C-G base editing is realized, providing a new idea for base editing.

[0005] The flexibility of base editing technology enables researchers to precisely modify the genomes of microorganisms, improve their traits to meet specific requirements and application scenarios, and enhance their application potential in the fields of industry, agriculture, medicine, and the environment. However, the commonly used Cas9 and base editing enzymes from mesophilic organisms lose their activity at high temperatures, and currently, there is a lack of enzymes that can perform base editing at high temperatures. Therefore, base editing technology has only been successfully applied in mesophilic microbial cells, and there are no reports on base editing technology for thermophilic microorganisms such as Methanolibacillus. Methanolibacillus is a microorganism that can efficiently utilize methanol raw materials at a high temperature of 50 °C and can be used for the biosynthesis of various products such as amino acids, which has important application value. However, due to the lack of available gene editing technology in Methanolibacillus, random mutagenesis is often used for strain improvement and screening, resulting in low breeding efficiency. Summary of the Invention

[0006] To address the above problems, the present invention provides a UNG mutant, a fusion protein, a base editing system, and an application that can be used for base editing of Methanolibacillus. The UNG mutant provided by the present invention can recognize the base C of Methanolibacillus and excise it to generate an AP site; the fusion protein provided by the present invention utilizes the targeting function of nThermoCas9 and the above function of the UNG mutant to enable editing of the base C in Methanolibacillus. The base editing system provided by the present invention expresses the fusion protein, and the base editing technology based on this base editing system can achieve precise editing of its genome by utilizing the DNA repair process of Methanolibacillus itself.

[0007] To achieve the above invention objectives, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a UNG mutant applicable to base editing of Methanolibacillus, and the amino acid sequence of the UNG mutant is as shown in SEQ ID NO.1.

[0009] This UNG mutant is obtained by mutating the endogenous wild-type UNG of Methanolibacillus. Since Methanolibacillus is a thermophilic bacterium, the UNG derived from Methanolibacillus is also heat-resistant and theoretically should have the ability to perform base editing on Methanolibacillus. However, through research, the present invention found that UNG cannot recognize C and cannot perform base editing on C. On the basis of the wild-type UNG, the present invention creatively mutates the 126th L-asparagine to L-aspartic acid to obtain a UNG mutant with the amino acid sequence as shown in SEQ ID NO.1. This mutant can recognize the C of Methanolibacillus and excise it to generate an AP site, thereby achieving base editing of C. This mutant fills the gap in the prior art that cannot perform base editing on Methanolibacillus and provides an efficient, precise, and feasible tool for gene editing of Methanolibacillus.

[0010] The mutant can be directly synthesized using the coding gene or obtained by PCR amplification method.

[0011] Exemplarily, the present invention provides a method for obtaining the above UNG mutant by PCR amplification, which is as follows:

[0012] Using the wild-type Methanosarcina barkeri MGA3 genome as a template, PCR amplification is carried out with the primer pairs UNG-R / N126-F with sequences as shown in SEQ ID NO.4 and SEQ ID NO.5 and the primer pair UNG-F / N126D-R with sequences as shown in SEQ ID NO.6 and SEQ ID NO.7. The PCR amplification system is:

[0013]

[0014] The reaction procedure is:

[0015]

[0016] The second aspect of the present invention provides the coding gene of the above UNG mutant.

[0017] Preferably, the nucleotide sequence of the coding gene is as shown in SEQ ID NO.2. Using this coding gene, the above UNG mutant can be directly obtained by synthesis method.

[0018] The third aspect of the present invention provides a fusion protein UNG N126D - nThermoCas9 , which is composed of the above UNG mutant and single-strand cleavage-inactivated nThermoCas9.

[0019] ThermoCas9 is derived from a thermophilic bacterium Geobacillus thermodenitrificans T12 , and can maintain its activity at a temperature of up to 70°C. This fusion protein utilizes the single-strand cleavage and positioning functions of nThermoCas9 and the function of the UNG mutant to excise C to form an AP site, and can also achieve base C editing in Methanosarcina barkeri at high temperature.

[0020] Preferably, the amino acid sequence of the fusion protein UNG N126D - nThermoCas9 is as shown in SEQ ID NO.3.

[0021] The fourth aspect of the present invention provides a base editing system that can be used for Methanosarcina barkeri. The base editing system is a plasmid expressing the above fusion protein UNG N126D - nThermoCas9 and gRNA.

[0022] The sequence of the gRNA is divided into two parts. One part is a sequence designed according to 20 bp of the target gene to be edited in Methanosarcina, and the other part is a fixed gRNA framework sequence. Those skilled in the art can design the sequence of the gRNA for the target gene to be edited in Methanosarcina through conventional techniques.

[0023] Preferably, the plasmid is plasmid pNW33N.

[0024] The fifth aspect of the present invention provides a method for constructing the above base editing system, specifically including the following operations:

[0025] Using plasmid PNW33N- ThermoCas9-gRNA as a template, the coding gene of nThermoCas9 and the plasmid backbone are amplified by PCR, and at the same time, the gRNA targeting the genome is introduced; the expression element of the gRNA and the coding gene of the linker of the fusion protein UNG N126D - nThermoCas9 are amplified by PCR; the coding gene of the UNG mutant is amplified by PCR;

[0026] The PCR amplification products are purified and ligated to construct the base editing system.

[0027] Preferably, the primers for amplifying the coding gene of nThermoCas9 by PCR are the primer pair T-F and T-R with sequences shown in SEQ ID NO.8 and SEQ ID NO.9.

[0028] Preferably, the primers for amplifying the plasmid backbone by PCR are primer A6 and primer PmtlR-R with sequences shown in SEQ ID NO.10 and SEQ ID NO.11.

[0029] Preferably, the primers for amplifying the coding gene of the linker by PCR are primer linker-f and primer linker-r with sequences shown in SEQ ID NO.14 and SEQ ID NO.15.

[0030] Preferably, the method for amplifying the coding gene of the UNG mutant by PCR is: using the genome of wild-type Methanosarcina MGA3 as a template, and amplifying with the primers UNG-R and primer N126-F with sequences shown in SEQ ID NO.4 and SEQ ID NO.5 and the primers UNG-F and primer N126D-R with sequences shown in SEQ ID NO.6 and SEQ ID NO.7.

[0031] The sixth aspect of the present invention provides the above-mentioned UNG mutants, encoding genes, fusion proteins UNG N126D - nThermoCas9 and the application of the base editing system in base editing of Methanosarcina thermophila.

[0032] Preferably, the base editing is the editing of base C.

[0033] The seventh aspect of the present invention provides a method for base editing of Methanosarcina thermophila: transferring the plasmid expressing the above-mentioned UNG mutant into the competent cells of Methanosarcina thermophila; or transferring the plasmid carrying the above-mentioned encoding gene into the competent cells of Methanosarcina thermophila; or transferring the plasmid expressing the above-mentioned fusion protein UNG N126D - nThermoCas9 into the competent cells of Methanosarcina thermophila; or transferring the above-mentioned base editing system into the competent cells of Methanosarcina thermophila.

[0034] Preferably, the base editing is the editing of base C.

[0035] Preferably, the method of transfer is electroporation.

[0036] Preferably, the method further includes culturing the competent cells of Methanosarcina thermophila that have completed the transfer in SOBsuc medium.

[0037] More preferably, the temperature of the culture is 50 ± 2 °C.

[0038] The beneficial effects of the present invention are as follows: The artificially designed endogenous UNG mutant of Methanosarcina thermophila provided by the present invention can glycosylate base C to generate an AP site, realizing the editing of base C. By using nThermoCas9 with activity at high temperature and this UNG mutant, the present invention constructs a base editing system expressing a fusion protein UNG N126D - nThermoCas9 and for the first time establishes a base editing technology in Methanosarcina thermophila. This base editing technology can achieve precise editing of its genome by utilizing the DNA repair process of Methanosarcina thermophila itself, providing an important enabling technology for the genetic transformation of Methanosarcina thermophila and having a breakthrough significance for the base editing of thermophilic Methanosarcina thermophila. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the plasmid map of plasmid PNW33N- ThermoCas9-gRNA in Example 3 of the present invention;

[0040] Figure 2 is the base editing system pNW33N-PmtlR- UNGN126D - nThermoCas9 - gRNA Plasmid map of

[0041] Figure 3 This is the base editing sequencing result based on UNG N126D - nThermoCas9 in Example 4 of the present invention. Detailed implementation manners

[0042] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the protection scope of the present invention.

[0043] Base editing technology uses the precise DNA positioning ability of the CRISPR / Cas9 system and the efficient catalytic ability of base deaminase for precise editing, which is more efficient than HDR. Since the commonly used Cas9 and base editing enzymes are from mesophilic organisms, base editing technology has currently only been successfully applied in mesophilic microbial cells and is not applicable to thermophilic microorganisms such as Methanolobus. Methanolobus has important application values, but lacks available gene editing technologies. Therefore, random mutagenesis is usually used for breeding at present, with low efficiency.

[0044] In the present invention, through the structural analysis of endogenous wild-type UNG in Methanolobus, the binding pocket of U in UNG was determined and the amino acids related to this binding pocket were mutated. It was found through research that when the N at the 126th position of wild-type UNG was mutated to D, this UNG mutant could specifically recognize and excise base C to form an AP site. Applying this UNG mutant to the base editing of Methanolobus MGA3 will solve the problem restricted by temperature. Based on this research result, the embodiments of the present invention provide a UNG mutant applicable to the base editing of Methanolobus, and its amino acid sequence is shown in SEQ ID NO.1.

[0045] The embodiments of the present invention also provide the coding gene of the above UNG mutant.

[0046] Based on this mutant, the embodiments of the present invention also provide a fusion protein composed of the above UNG mutant and single-strand cleavage-inactivated nThermoCas9 UNG N126D - nThermoCas9 .

[0047] The embodiments of the present invention also provide a base editing system that can be used for Bacillus methanolicus, and the base editing system is a plasmid expressing the above fusion protein UNG N126D - nThermoCas9 and gRNA.

[0048] The embodiments of the present invention also provide a construction method of the above base editing system.

[0049] The embodiments of the present invention also provide the above UNG mutant, encoding gene, fusion protein UNG N126D - nThermoCas9 and the application of the base editing system in base editing of Bacillus methanolicus, and provide a method for base editing of Bacillus methanolicus.

[0050] The following illustrates the solution of the present invention through specific embodiments.

[0051] The experimental materials used in the following examples:

[0052] Plasmid: Plasmid PNW33N was synthesized by Tsingke Biotechnology Co., Ltd.;

[0053] Cell line: Bacillus methanolicus MGA3 was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ), and Escherichia coli competent Trans1-T1 was a commercially available product;

[0054] Medium formula:

[0055] TSB medium: Tryptone 15 g / L, Soybean peptone 5 g / L, NaCl 5 g / L, adjust the pH to 7.0, sterilize at 121 °C for 20 min

[0056] LB liquid medium: Tryptone 10 g / L, Yeast extract powder 5 g / L, NaCl 10 g / L, sterilize at 121 °C for 20 min;

[0057] LB solid medium: Tryptone 10 g / L, Yeast extract powder 5 g / L, NaCl 10 g / L, 2% agar powder, sterilize at 121 °C for 20 min;

[0058] SOBsuc medium: Tryptone 20 g / L, Yeast extract 5 g / L, NaCl 0.5 g / L, KCl 0.2 g / L, MgCl2 1 g / L, MgSO4·7H2O 2.5 g / L, Sucrose 85.5 g / L;

[0059] Reagents: Primers were synthesized by Tsingke Biological Technology Co., Ltd.; High-fidelity DNA polymerase and DNA ligase were purchased from TransGen Biotech Co., Ltd.; DNA purification kit, plasmid mini-prep kit, and genomic DNA extraction kit were purchased from Tiangen Biochemical Technology Co., Ltd.

[0060] Other reagents or instruments used in the following examples, unless otherwise specified, were all conventional commercially available products obtained through commercial channels. The methods used in the following examples, unless otherwise specified, were all conventional methods in the art.

[0061] Example 1

[0062] This example provides a UNG mutant (named UNG N126D ) applicable to base editing of Bacillus methanolicus, and its amino acid sequence is shown in SEQ ID NO.1.

[0063] The UNG provided in this example N126D can be directly synthesized using the coding gene shown in SEQ ID NO.2, or can be obtained by the following method:

[0064] Using the wild-type Bacillus methanolicus MGA3 genome as a template, PCR amplification was performed with the primer pairs UNG-R / N126-F with sequences shown in SEQ ID NO.4 and SEQ ID NO.5 and the primer pair UNG-F / N126D-R with sequences shown in SEQ ID NO.6 and SEQ ID NO.7. The PCR amplification system is shown in Table 1, and the reaction program is shown in Table 2.

[0065] Table 1 PCR amplification system

[0066]

[0067] Table 2 PCR reaction program

[0068]

[0069] Purify the PCR amplification product to obtain UNG N126D .

[0070] Example 2

[0071] This example provides a fusion protein UNG N126D - nThermoCas9 , which is obtained by connecting nThermoCas9 and UNG in Example 1 N126D through a linker, and its amino acid sequence is shown in SEQ ID NO.3.

[0072] The fusion protein provided in this exampleUNG N126D - nThermoCas9 It was directly synthesized by Beijing Tsingke Biotechnology Co., Ltd. after codon optimization.

[0073] Example 3

[0074] This example provides a base editing system pNW33N-PmtlR- UNG N126D - nThermoCas9 - gRNA , and its construction method is as follows:

[0075] Using plasmid PNW33N- ThermoCas9-gRNA (with the structure as Figure 1 shown) as a template, the coding gene of nThermoCas9 was amplified with primer pairs T-F / T-R with sequences as shown in SEQ ID NO.8 and SEQ ID NO.9, the plasmid backbone was amplified with primer pairs A6 / PmtlR-R with sequences as shown in SEQ ID NO.10 and SEQ ID NO.11, and at the same time, the gRNA targeting the genome was introduced; the expression element of the gRNA was amplified with primer pairs GJ-F / PTA-R with sequences as shown in SEQ ID NO.12 and SEQ ID NO.13. The coding gene of the fusion protein UNG N126D - nThermoCas9 linker was amplified with primer pairs linker-f / linker-r with sequences as shown in SEQ ID NO.14 and SEQ ID NO.15. Using the wild-type Methanosarcina barkeri MGA3 genome as a template, the coding gene of the UNG mutant was amplified with primer pairs UNG-R / N126-F with sequences as shown in SEQ ID NO.4 and SEQ ID NO.5 and primer pairs UNG-F / N126D-R with sequences as shown in SEQ ID NO.6 and SEQ ID NO.7.

[0076] The PCR amplification system was the same as Table 1 in Example 1, and the reaction program was the same as Table 2 in Example 1.

[0077] Purify the PCR amplification products, and use the p EASY -Uni Seamless Cloning and Assembly Kit for multi-fragment ligation to construct nThermoCas9 -UNG fusion protein expression plasmid. The ligation system is shown in Table 3, and the ligation reaction conditions are 50°C for 15 min, and then cooled to 4°C.

[0078] Table 3 Ligation reaction system

[0079]

[0080] The obtained plasmid was transformed into Trans1-T1 Escherichia coli competent cells, and the monoclonal with correct sequencing was inoculated into liquid medium to extract the plasmid, thus obtaining pNW33N-PmtlR- UNG N126D - nThermoCas9 - gRNA 。 Its structure is as shown in Figure 2 and the nucleotide sequence is as shown in SEQ ID NO.16.

[0081] Comparative Example 1

[0082] This comparative example provided a base editing system pNW33N-PmtlR- UNG - nThermoCas9 - gRNA , and its construction method was basically the same as that of Example 3, except that the wild-type UNG was amplified with the primer pair UNG-F / UNG-R.

[0083] Comparative Example 2

[0084] This comparative example constructed a series of base editing systems based on enzymes and mutants from mesophilic organisms such as SpCas9 from Streptococcus pyogenes, FnCas12a from Francisella novicida, AID from eel, human AID (hAID), and UGI, a glycosylase inhibitor protein from Bacillus subtilis, namely: pNW33N- nSpCas9 - AID , pNW33N- dSpCas9 - AID , pNW33N- nSpCas9 - AID - UGI , pNW33N- dSpCas9 - AID - UGI , pNW33N- dFnCas12a - AID , pNW33N- dFnCas12a - hAID , pNW33N- dThermoCas9 - AID . The construction method was basically the same as that of Example 3, except that the primers used were primers routinely designed for the above-mentioned enzymes and mutants.

[0085] Example 4

[0086] This example provided pNW33N-PmtlR- constructed in Example 3 UNGN126D - nThermoCas9 - gRNA Application in base editing of Methanosarcina MGA3. The specific steps are as follows:

[0087] ① Take 50 μl of the preserved wild-type Methanosarcina MGA3 glycerol bacterial suspension and inoculate it into a 100 ml conical flask containing 5 ml of TSB medium (added with 1% anhydrous methanol). Culture it on a shaker at 50 °C and 220 rpm for about 12 h until the OD of the bacterial solution 600 is about 7 - 8;

[0088] ② Inoculate the bacterial solution obtained in step ① into 5 ml of pre-warmed SOBsuc medium at 50 °C (added with 1% anhydrous methanol), and control the initial OD 600 to be 0.2. Culture it on a shaker at 50 °C and 220 rpm until the OD of the bacterial solution 600 is about 4 - 4.5;

[0089] ③ Inoculate the bacterial solution obtained in step ② into 100 ml of pre-warmed SOBsuc medium at 50 °C (added with 1% anhydrous methanol), and control the initial OD 600 to be 0.05. Culture it on a shaker at 50 °C and 220 rpm until the OD 600 is 0.5;

[0090] Dispense all the bacterial solution into two sterile 50 ml centrifuge tubes, let it stand on ice for 15 min, and centrifuge at 4 °C (3000×g, 5 min). Wash the bacterial pellet collected from every 100 ml of bacterial solution with 20 ml of 1 mM HEPES buffer, and finally resuspend it with 0.4 ml of EP6000 buffer. Dispense it into sterile 1.5 ml EP tubes, 100 μl per tube, and divide it into 6 tubes;

[0091] Take 100 μl of Methanosarcina MGA3 competent cells and mix them with 2 μg of PNW33N - PmtlR - UNG N126D - nThermoCas9 - gRNA plasmid DNA, and incubate on ice for 5 min;

[0092] Transfer the mixture to a pre-cooled electroporation cuvette (0.2 cm electrode gap), and use an electroporator (2.5 kV) to expose the cells to a single electrical pulse;

[0093] The electroporated cells are cultured in a shaker flask with 5 ml of SOBsuc medium (added with 1% anhydrous methanol) for 12 h. Take 500 μl and spread it on an SOBsuc plate containing 5 μg / ml chloramphenicol, and culture it inverted at 50 °C for 36 h;

[0094] Pick monoclonal MGA3 for PCR verification. Inoculate the monoclonal with correct band size into SOBsuc liquid medium, add 1% anhydrous methanol, 5 μg / ml chloramphenicol and 2 mM mannitol for two generations of induction. Take the bacterial liquid for PCR amplification of the genomic fragment targeted by gRNA and send it for sequencing.

[0095] As Figure 3 shown, the sequencing results show that the base of the gRNA-targeted strand has changed, with base C changed to base A, indicating that the base editing of Bacillus methanolicus has been successfully achieved in the present invention.

[0096] Using the base editing systems of Comparative Example 1 and Comparative Example 2 to perform base editing on Bacillus methanolicus in the same method, no base editing was generated in the gRNA-targeted sequence. It is speculated that the reason why the base editing system of Comparative Example 2 did not generate base editing may be that the high-temperature growth conditions of Bacillus methanolicus inhibited the activity of the enzyme. The wild-type UNG is heat-resistant, but the base editing system expressing the fusion protein of wild-type UNG and nThermoCas9 in Comparative Example 1 also did not achieve base editing in Bacillus methanolicus. The reason may be that the wild-type UNG cannot recognize C.

[0097] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements or improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A UNG mutant applicable to base editing of Bacillus methanolicus, characterized in that: The amino acid sequence of the UNG mutant is shown in SEQ ID NO.

1.

2. The coding gene of the UNG mutant according to claim 1.

3. A fusion protein UNG N126D - nThermoCas9 , characterized in that, The fusion protein is composed of the UNG mutant of claim 1 and the single-stranded cleavage-inactivated nThermoCas9. UNG N126D - nThermoCas9 The amino acid sequence is shown in SEQ ID NO.

3.

4. A base editing system that can be used for Bacillus methanolicus, characterized in that: The base editing system is for expressing the fusion protein of claim 3 UNG N126D - nThermoCas9 and gRNA plasmids.

5. The base editing system according to claim 4, characterized in that The backbone vector of the plasmid is plasmid pNW33N.

6. The method for constructing a base editing system according to claim 4 or 5, characterized in that: The specific operations include the following: using plasmid PNW33N- ThermoCas9-gRNA As a template, the coding gene and plasmid backbone of nThermoCas9 were amplified by PCR, and the gRNA targeting the genome was introduced at the same time; the expression element of the gRNA and the fusion protein were amplified by PCR UNG N126D - nThermoCas9 The gene encoding the linker; amplifying the coding gene of the UNG mutant by PCR; The PCR amplification products are purified, connected, and the base editing system is constructed.

7. The UNG mutant of claim 1, the encoding gene of claim 2, and the fusion protein of claim 3 UNG N126D - nThermoCas9 and use of the base editing system according to claim 4 or 5 in base editing of Bacillus methanolica, characterized in that The base editing is to change base C to base A.

8. A method for base editing of Bacillus methanolicus, characterized in that: The plasmid expressing the UNG mutant of claim 1 is transferred into the competent cells of Bacillus methanolicus; or the plasmid carrying the coding gene of claim 2 is transferred into the competent cells of Bacillus methanolicus; or the plasmid expressing the fusion protein of claim 3 is transferred into the competent cells of Bacillus methanolicus. UNG N126D - nThermoCas9 The plasmid is transferred into methanolic Bacillus competent cells; or the base editing system described in claim 4 or 5 is transferred into methanolic Bacillus competent cells; the base editing is to change base C to base A.

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