UNG mutant capable of being used for base editing of bacillus methanolicus, fusion protein, base editing system and application

Abstract

The invention belongs to the technical field of base editing, and particularly relates to a UNG mutant capable of being used for base editing of bacillus methanolicus, a fusion protein, a base editing system and application. The amino acid sequence of the UNG mutant provided by the invention is as shown in SEQ ID NO. 1. The UNG mutant can recognize a base C of bacillus methylophilus and perform excision to generate an AP site, so that base editing of the base C is realized. The fusion protein provided by the invention is composed of the UNG mutant and nThermoCas9 which is inactivated by single-chain cleavage, and a basic group C in the bacillus methylophilus can be edited into a basic group T at a high temperature. The base editing system provided by the invention expresses the fusion protein and the gRNA, and a base editing technology based on the base editing system provides an important enabling technology for genetic modification of bacillus methylophilus, and has breakthrough significance for base editing of thermophilic bacillus methylophilus.

Description

Technical Field

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

[0002] For a long time, precise gene editing has required the help of homologous recombination (HDR), but HDR is inefficient in many cells. At the same time, most HDRs require the use of Cas9 to produce DNA double-strand breaks to improve efficiency, and the inevitable non-homologous end joining (NHEJ) will cause random mutations. These two shortcomings of HDR have severely limited the development of the field of precise gene editing. In addition, HDR and NEHJ are both endogenous DNA repair mechanisms of cells, which are not convenient for rational transformation.

[0003] Base editing technology came out in 2016. This technology uses the CRISPR / Cas9 system's precise DNA positioning capabilities and the efficient catalytic ability of base deaminases to perform precise editing. Compared with HDR-based gene editing technology, base editing technology does not rely on inefficient DNA repair processes such as HDR, and does not use Cas9 to cause double-strand breaks in DNA. It will not induce random mutations caused by NHEJ, so it is usually easier to implement and has higher efficiency.

[0004] The technologies that have been made public so far include CBE, ABE, and GBE technologies. CBE and ABE technologies rely on the DNA positioning ability of the CRISPR / Cas9 system. By fusing the Cas9 protein with an inactivated double-stranded DNA cutting function and cytosine (C) or adenine (A) deaminase, cytosine deaminase or adenine deaminase is positioned at a specific position in the genome, catalyzing the deamination reaction of C or A at a specific position, converting it into uracil (U) or hypoxanthine (I), which is then recognized as thymine (T) or guanine (G) during DNA replication, achieving the editing of CT bases and AG bases, respectively. GBE technology was made public 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 produce an apurinic / apyrimidinic (AP) site, and CG base editing is achieved through DNA repair, providing a new idea for base editing.

[0005] The flexibility of base editing technology allows researchers to precisely modify the genomes of microorganisms and improve their traits to meet specific needs and application scenarios, thereby increasing their potential for application in industry, agriculture, medicine, and the environment. However, the commonly used Cas9 and base editing enzymes from mesophilic organisms lose their activity at high temperatures. There is currently a lack of enzymes that can perform base editing at high temperatures, so base editing technology has only been successfully applied in mesophilic microbial cells, and there have been no reports of base editing technology for thermophilic microorganisms such as methanolic Bacillus. Methanolic Bacillus is a microorganism that can efficiently utilize methanol raw materials at a high temperature of 50°C. It can be used for the biosynthesis of a variety of products such as amino acids and has important application value. However, due to the lack of available gene editing technology for methanolic Bacillus, random mutagenesis is often used to improve and screen strains, and the breeding efficiency is low. Summary of the invention

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

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical scheme: In a first aspect, the present invention provides a UNG mutant that can be used for base editing of Bacillus methanolicus, and the amino acid sequence of the UNG mutant is shown in SEQ ID NO.1.

[0008] The UNG mutant is obtained by mutating the endogenous wild-type UNG of Bacillus methanolica. Bacillus methanolica is a thermophilic bacterium, so the UNG derived from Bacillus methanolica is also resistant to high temperatures, and theoretically should have the ability to perform base editing on Bacillus methanolica. However, the present invention has found through research that UNG cannot recognize C and cannot perform base editing on C. On the basis of wild-type UNG, the present invention creatively mutates L-asparagine at position 126 to L-aspartic acid to obtain a UNG mutant with an amino acid sequence as shown in SEQ ID NO.1. The mutant can recognize C of Bacillus methanolica and excise it to produce an AP site, thereby achieving base editing of C. This mutant fills the gap that the prior art cannot perform base editing on Bacillus methanolica, and provides an efficient, accurate and feasible tool for gene editing of Bacillus methanolica.

[0009] The mutant can be directly synthesized using the coding gene, or can be obtained by PCR amplification.

[0010] Exemplarily, the present invention provides a method for obtaining the above-mentioned UNG mutant by PCR amplification, which is as follows: The wild-type methanolic Bacillus MGA3 genome was used as a template, and PCR amplification was performed using the primer pair UNG-R / N126-F shown in SEQ ID NO.4 and SEQ ID NO.5 and the primer pair UNG-F / N126D-R shown in SEQ ID NO.6 and SEQ ID NO.7. The PCR amplification system was:

[0011] The reaction procedure is:

[0012] The second aspect of the present invention provides a gene encoding the above-mentioned UNG mutant.

[0013] Preferably, the nucleotide sequence of the coding gene is shown in SEQ ID NO. 2. The coding gene can be used to directly obtain the above UNG mutant by synthetic method.

[0014] The third aspect of the present invention provides a fusion protein UNG N162D - nThermoCas9 , which consists of the above-mentioned UNG mutant and nThermoCas9 inactivated for single-stranded cleavage.

[0015] ThermoCas9 is derived from thermophilic bacteria Geobacillus thermodenitrificans T12 , and can maintain activity at temperatures as high as 70°C. This fusion protein utilizes the single-strand cutting and positioning functions of nThermoCas9 and the function of the UNG mutant to remove C to form an AP site, and can also edit the base C in methanolic Bacillus at high temperatures.

[0016] Preferably, the fusion protein UNG N162D - nThermoCas9 The amino acid sequence is shown in SEQ ID NO.3.

[0017] The fourth aspect of the present invention provides a base editing system that can be used for methanolic Bacillus, wherein the base editing system is for expressing the above-mentioned fusion protein UNG N162D - nThermoCas9 and gRNA plasmids.

[0018] 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 methanolic Bacillus, 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 methanolic Bacillus by conventional techniques.

[0019] Preferably, the plasmid is plasmid pNW33N.

[0020] The fifth aspect of the present invention provides a method for constructing the above-mentioned base editing system, which specifically includes the following operations: 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 N162D - nThermoCas9 The coding gene of 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.

[0021] Preferably, the primers for amplifying the gene encoding the nThermoCas9 by PCR are primer pairs TF and TR whose sequences are shown in SEQ ID NO.8 and SEQ ID NO.9.

[0022] Preferably, the primers for amplifying the plasmid backbone by PCR are primer A6 and primer PmtlR-R, whose sequences are shown in SEQ ID NO.10 and SEQ ID NO.11.

[0023] Preferably, the primers for amplifying the gene encoding the linker by PCR are primer linker-f and primer linker-r, whose sequences are shown in SEQ ID NO.14 and SEQ ID NO.15.

[0024] Preferably, the method for amplifying the encoding gene of the UNG mutant by PCR is: using the wild-type methanolic Bacillus MGA3 genome as a template, and amplifying with primers UNG-R and primer N126-F as shown in SEQ ID NO.4 and SEQ ID NO.5, and primers UNG-F and primer N126D-R as shown in SEQ ID NO.6 and SEQ ID NO.7.

[0025] The sixth aspect of the present invention provides the above-mentioned UNG mutant, encoding gene, fusion protein UNG N162D - nThermoCas9 and application of base editing system in base editing of Bacillus methanolicus.

[0026] Preferably, the base editing is editing of base C.

[0027] The seventh aspect of the present invention provides a method for base editing of methanolic Bacillus: transferring a plasmid expressing the above-mentioned UNG mutant into a methanolic Bacillus competent cell; or transferring a plasmid carrying the above-mentioned encoding gene into a methanolic Bacillus competent cell; or transferring a plasmid expressing the above-mentioned fusion protein into a methanolic Bacillus competent cell. UNG N162D - nThermoCas9 The plasmid is transferred into methanolic Bacillus competent cells; or the above-mentioned base editing system is transferred into methanolic Bacillus competent cells.

[0028] Preferably, the base editing is editing of base C.

[0029] Preferably, the transfer method is electroporation.

[0030] Preferably, the method further comprises culturing the transferred Bacillus methanolica competent cells in SOBsuc medium.

[0031] More preferably, the culture temperature is 50±2°C.

[0032] The beneficial effect of the present invention is that the artificially designed endogenous UNG mutant of Bacillus methanolicus provided by the present invention can glycosylate base C to generate AP site, thereby realizing the editing of C base. The present invention constructs a fusion protein expression system by using nThermoCas9 which is active at high temperature and the UNG mutant. UNG N162D - nThermoCas9 The base editing system established the base editing technology in Bacillus methanolica for the first time. This base editing technology utilizes the DNA repair process of Bacillus methanolica to achieve precise editing of its genome, providing an important enabling technology for the genetic transformation of Bacillus methanolica and having groundbreaking significance for base editing of thermophilic Bacillus methanolica. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The plasmid PNW33N- ThermoCas9-gRNA Plasmid map of Figure 2 The base editing system pNW33N-PmtlR- UNG N162D - nThermoCas9 - gRNA Plasmid map of Figure 3 In Example 4 of the present invention, UNG N162D - nThermoCas9 Base editing sequencing results. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with 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. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0035] Base editing technology uses the DNA precision positioning ability of the CRISPR / Cas9 system and the efficient catalytic ability of base deaminases for precise editing, which is more efficient than HDR. Since the commonly used Cas9 and base editing enzymes come from mesophilic organisms, base editing technology has only been successfully applied in mesophilic microbial cells and is not suitable for thermophilic microorganisms such as methanolic Bacillus. Methanolic Bacillus has important application value, but there is a lack of available gene editing technology, so random mutagenesis is usually used for breeding at this stage, which is inefficient.

[0036] The present invention determines the binding pocket of U in UNG and mutates the amino acids related to the binding pocket by structural analysis of endogenous wild-type UNG of Bacillus methanolicus. It is found that when the N at position 126 of wild-type UNG is mutated to D, the UNG mutant can specifically recognize and excise the base C to form an AP site. Applying the UNG mutant to base editing of Bacillus methanolicus MGA3 will solve the problem of temperature limitation. Based on the research results, the embodiment of the present invention provides a UNG mutant that can be applied to base editing of Bacillus methanolicus, and its amino acid sequence is shown in SEQ ID NO.1.

[0037] The embodiments of the present invention also provide a gene encoding the above UNG mutant.

[0038] Based on this mutant, the present invention also provides a fusion protein consisting of the above-mentioned UNG mutant and the nThermoCas9 inactivated by single-stranded cleavage. UNG N162D - nThermoCas9 .

[0039] The present invention also provides a base editing system that can be used for methanolic Bacillus. The base editing system is used to express the above fusion protein UNG N162D - nThermoCas9 and gRNA plasmids.

[0040] An embodiment of the present invention also provides a method for constructing the above-mentioned base editing system.

[0041] The present invention also provides the above-mentioned UNG mutant, encoding gene, fusion protein UNG N162D - nThermoCas9 and application of the base editing system in base editing of Bacillus methanolicus, and a method for base editing of Bacillus methanolicus is provided.

[0042] The scheme of the present invention is described below through specific embodiments.

[0043] The experimental materials used in the following examples are: Plasmid: Plasmid PNW33N was synthesized by Qingke Biotechnology Co., Ltd. Cell line: Bacillus methanolicus MGA3 was purchased from German Microbiological Culture Collection DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen), and Escherichia coli competent strain Trans1-T1 was commercially available; Culture medium formula: TSB medium: tryptone 15 g / L, soy peptone 5 g / L, NaCl 5 g / L, adjust pH to 7.0, sterilize at 121℃ for 20 min LB liquid medium: tryptone 10 g / L, yeast extract powder 5 g / L, NaCl 10 g / L, high temperature sterilization at 121℃ for 20 min; LB solid medium: tryptone 10 g / L, yeast extract powder 5 g / L, NaCl 10 g / L, 2% agar powder, sterilized at 121°C for 20 min; 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; Reagents: Primers were synthesized from Qingke Biotechnology Co., Ltd.; high-fidelity DNA polymerase and DNA ligase were purchased from Quanshijin Biotechnology Co., Ltd.; DNA purification kit, plasmid mini-extraction kit, and genomic DNA extraction kit were purchased from Tiangen Biochemical Technology Co., Ltd.

[0044] Unless otherwise specified, other reagents or instruments used in the following examples are conventional commercial products obtained from commercial channels. Unless otherwise specified, the methods used in the following examples are conventional methods in the art.

[0045] Example 1 This example provides a UNG mutant (named UNG N162D ), whose amino acid sequence is shown in SEQ ID NO.1.

[0046] The UNG provided in this embodiment N162D It can be directly synthesized using the coding gene shown in SEQ ID NO.2, or it can be obtained by the following method: Using the wild-type methanolic Bacillus MGA3 genome as a template, PCR amplification was performed using the primer pair UNG-R / N126-F shown in SEQ ID NO.4 and SEQ ID NO.5 and the primer pair UNG-F / N126D-R shown in SEQ ID NO.6 and SEQ ID NO.7. The PCR amplification system is shown in Table 1, and the reaction procedure is shown in Table 2.

[0047] Table 1 PCR amplification system

[0048] Table 2 PCR reaction procedure

[0049] The PCR amplification product was purified to obtain UNG N162D .

[0050] Example 2 This embodiment provides a fusion protein UNG N162D - nThermoCas9 The fusion protein is a combination of nThermoCas9 and UNG in Example 1. N162D It is obtained by connecting through a linker, and its amino acid sequence is shown in SEQ ID NO.3.

[0051] The fusion protein provided in this example UNG N162D - nThermoCas9 It was directly synthesized by Beijing Qingke Biotechnology Co., Ltd. after codon optimization.

[0052] Example 3 This embodiment provides a base editing system pNW33N-PmtlR- UNG N162D - nThermoCas9 - gRNA , and its construction method is as follows: Plasmid PNW33N- ThermoCas9-gRNA (Structure such as Figure 1As shown in SEQ ID NO.8 and SEQ ID NO.9, the coding gene of nThermoCas9 was amplified using the primer pair TF / TR as shown in SEQ ID NO.8 and SEQ ID NO.9, the plasmid backbone was amplified using the primer pair A6 / PmtlR-R as shown in SEQ ID NO.10 and SEQ ID NO.11, and the gRNA targeting the genome was introduced at the same time; the expression element of gRNA was amplified using the primer pair GJ-F / PTA-R as shown in SEQ ID NO.12 and SEQ ID NO.13. The fusion protein was amplified using the primer pair linker-f / linker-r as shown in SEQ ID NO.14 and SEQ ID NO.15 UNG N162D - nThermoCas9 The coding gene of the linker is amplified using the wild-type methanolic Bacillus MGA3 genome as a template and the primer pair UNG-R / N126-F as shown in SEQ ID NO.4 and SEQ ID NO.5 and the primer pair UNG-F / N126D-R as shown in SEQ ID NO.6 and SEQ ID NO.7.

[0053] The PCR amplification system is the same as that in Table 1 of Example 1, and the reaction procedure is the same as that in Table 2 of Example 1.

[0054] The PCR amplification product was purified and EASY -Uni Seamless Cloning and Assembly Kit for multi-fragment ligation and construction 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 reduced to 4°C.

[0055] Table 3 Ligation reaction system

[0056] The obtained plasmid was transformed into Trans1-T1 Escherichia coli competent cells, and the single clone with correct sequencing was inoculated into liquid culture medium, and the plasmid was extracted to obtain pNW33N-PmtlR- UNG N162D - nThermoCas9 - gRNA Its structure is as follows Figure 2 The nucleotide sequence is shown in SEQ ID NO.16.

[0057] Comparative Example 1 This comparative example provides a base editing system pNW33N-PmtlR- UNG - nThermoCas9 - gRNA The construction method is basically the same as that of Example 3, except that the wild-type UNG is amplified using primer pair UNG-F / UNG-R.

[0058] Comparative Example 2 In this comparative example, a series of base editing systems were constructed based on nucleases such as SpCas9 from Streptococcus pyogenes, FnCas12a from Francisella novicida, AID from eel, human AID (hAID), glycosylase inhibitor protein UGI from Bacillus subtilis and other mesophilic enzymes and mutants, 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 is basically the same as that of Example 3, except that the primers used are conventionally designed primers for the above enzyme and mutant.

[0059] Example 4 This example provides pNW33N-PmtlR- UNG N162D - nThermoCas9 - gRNA Application in base editing of Bacillus methanolicus MGA3. The specific steps are as follows: ① Take 50 μl of the glycerol suspension of wild-type methanolic Bacillus MGA3 and inoculate it into a 100 ml conical flask containing 5 ml TSB medium (added with 1% anhydrous methanol). Incubate at 50°C and 220 rpm for about 12 h until the OD of the bacterial solution reaches 600 About 7~8; ②Inoculate the bacterial solution obtained in step ① into 5 ml SOBsuc medium (added with 1% anhydrous methanol) preheated at 50°C to control the initial OD 600 The bacterial solution was cultured at 50°C and 220 rpm in a shaking incubator until the OD 600 About 4~4.5; ③Inoculate the bacterial solution obtained in step ② into 100 ml SOBsuc medium (added with 1% anhydrous methanol) preheated at 50°C to control the initial OD 600 The culture was incubated at 50°C and 220 rpm in a shaking incubator until the OD 600 is 0.5; The entire bacterial solution was divided into two sterile 50 ml centrifuge tubes, placed on ice for 15 min, centrifuged at 4°C (3000×g, 5 min), and the collected bacterial sludge was washed with 20 ml 1mM HEPES buffer for every 100 ml bacterial solution, and finally resuspended with 0.4 ml EP6000 buffer and divided into 6 sterile 1.5 ml EP tubes, 100 μl per tube; Take 100 μl methanolic Bacillus MGA3 competent cells and 2 μg PNW33N-PmtlR- UNG N162D - nThermoCas9 - gRNA The plasmid DNA was mixed and incubated on ice for 5 min; The mixture was transferred to a pre-chilled electroporation cuvette (0.2 cm electrode gap), and the cells were exposed to a single electric pulse using an electroporator (2.5 kV); The electrotransfected cells were cultured in 5 ml SOBsuc medium (supplemented with 1% anhydrous methanol) for 12 h in a shaking flask, and 500 μl was spread on a SOBsuc plate containing 5 μg / ml chloramphenicol and incubated at 50°C for 36 h; The MGA3 monoclone was picked for PCR verification, and the monoclone with the correct band size was inoculated into SOBsuc liquid culture medium, induced for two generations by adding 1% anhydrous methanol, 5 μg / ml chloramphenicol and 2 mM mannitol. The bacterial liquid was used to PCR amplify the genomic fragment targeted by gRNA and sent for sequencing.

[0060] like Figure 3 As shown, the sequencing results show that the gRNA targeting chain has undergone a base change, and base C is changed to base A, indicating that the present invention successfully achieves base editing of Bacillus methanolicus.

[0061] The base editing systems of Comparative Examples 1 and 2 were used to perform base editing on methanolic Bacillus in the same way, and no base editing was produced in the gRNA targeting sequence. It is speculated that the reason why the base editing system of Comparative Example 2 did not produce base editing may be that the high temperature growth conditions of methanolic Bacillus inhibited the activity of the enzyme. Wild-type UNG is resistant to high temperatures, 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 methanolic Bacillus, and the reason may be that wild-type UNG cannot recognize C.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should 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 N162D - nThermoCas9 , characterized in that, It is composed of the UNG mutant described in claim 1 and the nThermoCas9 inactivated by single-stranded cleavage.

4. The fusion protein according to claim 3 UNG N162D - nThermoCas9 , characterized in that, The fusion protein UNG N162D - nThermoCas9 The amino acid sequence is shown in SEQ ID NO.

3.

5. A base editing system that can be used in Bacillus methanolicus, characterized in that: The base editing system is to express the above fusion protein UNG N162D - nThermoCas9 and gRNA plasmids.

6. The base editing system according to claim 5, characterized in that The plasmid is plasmid pNW33N.

7. The method for constructing a base editing system according to claim 5 or 6, 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 N162D - 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.

8. The UNG mutant according to claim 1, the encoding gene according to claim 2, or the fusion protein according to claim 3 or 4 UNG N162D - nThermoCas9 And the use of the base editing system described in claim 5 or 6 in base editing of Bacillus methanolicus.

9. The use according to claim 8, wherein the base editing is editing of base C.

10. 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 or 4 is transferred into the competent cells of Bacillus methanolicus. UNG N162D - nThermoCas9 The plasmid is transferred into methanolic Bacillus competent cells; or the base editing system described in claim 5 or 6 is transferred into methanolic Bacillus competent cells.

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