Cytosine deaminase and related biological material and application thereof

Abstract

The invention discloses cytosine deaminase as well as a related biological material and application thereof, and belongs to the technical field of protein. The technical problem to be solved by the invention is to excavate natural cytidine deaminase without sequence preference and improve the base editing efficiency and range. The invention provides a 15th-143rd site protein of which the amino acid sequence is SEQ ID NO.2. The invention also provides a preparation method of the protein. The invention also provides application of the protein and related biological materials thereof in single base editing. According to the application, metagenome big data is utilized, comprehensive and systematic information mining analysis is carried out on novel cytidine deaminase, and a protein L85 which is highly similar to a known DddA protein structure but low in sequence similarity is selected from the novel cytidine deaminase for functional verification. The protein L85 has high catalytic activity as cytidine deaminase, is not limited by target sequence preference, can be widely applied to gene editing in vitro and in cells / tissues, and has good application potential and development value.

Description

Technical Field

[0001] The present application belongs to the field of protein technology, and specifically relates to cytosine deaminase and related biomaterials and applications. Background Art

[0002] Genome editing technology is a genetic engineering technology that uses sequence-specific nucleases to modify specific locations in the genome of an organism. The process is to generate double-strand breaks (DSBs) in the genome, thereby stimulating the cell's endogenous repair mechanism, such as non-homologous end joining repair or homologous recombination repair, to perform site-specific artificial modifications such as knockout, replacement, and insertion of target sites in the genome. At present, commonly used genome editing technologies include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR / Cas). Compared with the first two gene editing technologies, CRISPR / Cas technology is more accurate, convenient, and efficient.

[0003] Single-nucleotide polymorphism (SNP) is the genetic basis of agronomic traits of crops. Changes in many important crop traits are usually caused by variations in a single base. At present, new base editing systems for targeted gene site-directed modification based on the CRISPR system, such as the cytosine base editing system and the adenine base editing system, have been developed and widely used in a variety of organisms such as plants and animals. Cytidine deaminases that have been successfully used in plant base editing systems include APOBEC1, APOBEC3, DddA, etc., and adenosine deaminases include TadA7.10, TadA8e, etc.

[0004] Due to the toxicity of DddA cytidine deaminase, the DddA deaminase-based cytosine base editing system mainly uses two different Cas9s or two TALENs to combine with DddA split into two halves to form a fusion protein. Under the guidance of sgRNA, the two halves will only restore catalytic activity when they bind to the targeted gene region, deaminate the C base in the editing window into dU, and finally achieve base editing of the targeted gene CG to TA in the organelles and nuclei through DNA replication and repair. However, in the organelles or nuclei of corn, the DddA deaminase-based cytosine base editing technology still has the disadvantage of low editing efficiency. Summary of the invention

[0005] The purpose of this application is to explore proteins with natural non-sequence preferences and improve the efficiency and scope of base editing. Specifically, the technical problem to be solved by this application is how to improve the editing efficiency and scope of cytosine base editors.

[0006] In order to solve the above technical problems, the present application provides a protein, which may be any of the following:

[0007] A1) a protein whose amino acid sequence is SEQ ID NO. 2;

[0008] A2) a protein whose amino acid sequence is positions 15 to 143 of SEQ ID NO. 2;

[0009] A3) a protein related to deaminase having an amino acid sequence represented by A1) or A2) with substitution and / or deletion and / or addition of amino acid residues, having 80% or more identity with the protein represented by A1) or A2);

[0010] A4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1), A2) or A3).

[0011] Furthermore, the connection described in A4) can be via a peptide bond.

[0012] Furthermore, the tags described herein include, but are not limited to: GST (glutathione sulfhydryltransferase) tag protein, His tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0013] In some embodiments of the present application, the tag is a His tag.

[0014] The present application also provides a biological material related to the above-mentioned protein, and the biological material may be any one of the following:

[0015] B1) a nucleic acid molecule encoding the above protein;

[0016] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0017] B3) a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2);

[0018] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0019] B5) a transgenic plant cell line containing the nucleic acid molecule described in B1), a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3);

[0020] B6) transgenic plant tissue containing the nucleic acid molecule described in B1), transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3);

[0021] B7) A transgenic plant organ containing the nucleic acid molecule described in B1), a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3).

[0022] Furthermore, in the biological material, the nucleic acid molecule in B1) may be at least one of the following:

[0023] g1) the coding sequence of the coding strand is a cDNA molecule or a DNA molecule shown in positions 2 to 388 of SEQ ID NO.1;

[0024] g2) A cDNA molecule or a DNA molecule that has 80% or more identity with the nucleotide sequence defined in g1) and encodes the protein.

[0025] In the present application, the expression cassette refers to a DNA capable of expressing the protein in a host cell (such as a microbial cell), which may include not only a promoter for initiating transcription of the protein gene, but also a terminator for terminating transcription of the protein gene. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present application include, but are not limited to, constitutive promoters, tissue, organ and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to, the Ubiquitin promoter of maize, the constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiology 120:979-992); the chemically inducible promoter from tobacco, pathogenesis-related protein 1 (PR1, induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); the tomato proteinase inhibitor II promoter (PIN2) or the LAP promoter (both of which can be induced by methyl jasmonate); heat shock promoters (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (China Patent No. 2007 10099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J. 4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in their entirety. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, e.g., Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

[0026] In some embodiments of the present application, the expression cassette utilizes T7 promoter to initiate transcription and translation of deaminase L85 and vector backbone sequence.

[0027] In the above, the recombinant microorganism can specifically be bacteria, yeast, algae and fungi.

[0028] In some embodiments of the present application, the bacterial liquid has the same meaning as "culture", and the term "culture" refers to a general term for a liquid or solid product (all substances in a culture container) with a microbial population after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms, or can contain a certain amount of culture medium, metabolites, or other components produced during the culture process. It can also be a mixture containing a certain amount of culture medium and microbial metabolites without the microbial cells.

[0029] The present application also provides the use of the above protein as cytidine deaminase.

[0030] The present application also provides the use of the above protein in the preparation of cytidine deaminase.

[0031] The present application also provides the use of the above-mentioned biological material in the preparation of cytidine deaminase.

[0032] The present application also provides the use of the above-mentioned protein in single-base editing.

[0033] The present application also provides the use of the above-mentioned protein in the preparation of single-base editing products.

[0034] The present application also provides the application of the above-mentioned biological materials in single-base editing.

[0035] The present application also provides the use of the above-mentioned biological materials in the preparation of single-base editing products.

[0036] In the present application, the single base editing may be cytosine base editing in cytosine deoxynucleoside (or cytosine nucleoside).

[0037] Furthermore, the cytosine base editing may be catalyzing the deamination of cytosine base to form uracil base.

[0038] In the present application, the product of the single-base editing may be a base editor. More specifically, the product of the single-base editing may be a cytosine base editor.

[0039] In the present application, the base editor may be a fusion protein containing cytidine deaminase, Cas protein and uracil glycosylase inhibitor, and the cytidine deaminase may be the above-mentioned protein.

[0040] Furthermore, the Cas protein may be nCas9.

[0041] Furthermore, the fusion protein is a protein formed by connecting the cytidine deaminase, the Cas protein, the uracil glycosylase inhibitor and the nuclear localization signal.

[0042] In the present application, the meaning of the cytidine deaminase is equivalent to cytosine deaminase.

[0043] The term "identity" as used herein refers to the sequence similarity between amino acid or nucleic acid sequences. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. The above 80% or more identity can be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity.

[0044] The 80% or more identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The 85% or more identity may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The 90% or more identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The greater than 95% identity may be at least 95%, 96%, 97%, 98% or 99% identity.

[0045] The present application also provides a method for deaminating cytosine, which may include using the above protein (as cytosine deaminase) to catalyze the deamination reaction of cytosine.

[0046] Furthermore, the cytosine in the method may be cytosine in a nucleic acid (nucleotide).

[0047] In some embodiments of the present application, the method comprises the following steps: placing the above-mentioned protein (as cytosine aminase) and substrate DNA in the same reaction system (deamination reaction system).

[0048] In some embodiments of the present application, the deamination reaction system is: 50 μL of the above protein (i.e., L85 protein (20 nM)) and 10 μL of a deamination buffer solution containing a DNA substrate are mixed. The deamination buffer solution containing a DNA substrate is composed of: 20 mM MES, 200 mM NaCl, 1 mM DTT, 8% Ficoll70, and 1 μM DNA substrate.

[0049] The deamination reaction conditions are as follows: subject the mixture to a deamination reaction at 37°C for 1 hour; then, 3 μL of UDG and 7 μL of 10×UDG buffer are added to the system, and the enzyme digestion reaction is carried out at 37°C for 30 minutes; 7 μL of 1M NaOH is added to the system, and the reaction is terminated at 95°C for 3 minutes. The obtained deamination reaction sample is used for subsequent electrophoresis verification.

[0050] The technical effects achieved by this application are as follows:

[0051] This application uses metagenomic big data to conduct a comprehensive and systematic bioinformatics mining analysis on new cytidine deaminases, and selects a protein L85 that is highly similar to the known DddA protein structure but has a low sequence similarity (identity <54.10%) for functional verification. As a cytidine deaminase, the protein L85 has high catalytic activity and no target sequence preference restrictions. It can be widely used in gene editing in vitro and in cells / tissues, and has good application potential and development value. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the evolutionary tree analysis of deaminase in each family.

[0053] Figure 2 Comparison of the novel deaminase and the known DddA protein structure. In the protein structure comparison results, yellow represents the novel deaminase and pink represents the known DddA protein. The RMSD value represents the root mean square deviation between the ligand structure and the reference structure. It is an indicator used to measure the structural similarity between two structures. The smaller the value, the more similar the structures. TM-Score is the template modeling score. The higher the score, the more similar the protein structures.

[0054] Figure 3 Schematic diagram of the deaminase purification system.

[0055] Figure 4 It is an analysis system for the deamination activity of deaminase.

[0056] Figure 5 These are the results of two validation tests of in vitro deamination activity.

[0057] Figure 6A DNA substrate for in vitro validation of target sequence preference.

[0058] Figure 7 Validate target sequence preference results for candidate proteins in vitro. DETAILED DESCRIPTION

[0059] The present application is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present application, not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvements by ordinary technicians in the technical field, and do not constitute a limitation of the present application in any way.

[0060] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0061] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0062] This application uses metagenomic big data to conduct a comprehensive and systematic bioinformatics mining analysis on new cytosine deaminases, and selects a protein L85 that is highly similar to the known DddA protein structure but has a low sequence similarity (identity <54.10%) for subsequent experimental verification. In vitro experiments show that the L85 protein is highly active and has no sequence preference, and can deaminize C of AC / TC / GC / CC.

[0063] Example 1: Novel cytidine deaminase mining process

[0064] 1.1. All sequenced metagenomic assembly nucleic acid sequence information was retrieved and downloaded from JGI and NCBI biological databases; protein sequences were annotated using Prodigal software to obtain all proteins; and domain-specific annotations were performed on the predicted coding genes using the Pfam database hidden Markov model.

[0065] 1.2. Because DddA belongs to the SCP1.201 family, its full length is >1400aa, and the real deamination function is the second half of the protein - DddAtox, whose annotation entry is SCP1.201-deam, so this entry is used as a keyword for screening; all the candidate proteins screened were aligned by Mafft software, and according to prior knowledge, the active site of DddAtox is glutamic acid E at position 1347, so proteins with the same site were screened; because DddA is involved in the type VI secretion system (T6SS) of the intercellular protein delivery system, and its downstream has the immune protein DddIA, the two interact to inhibit the toxicity of the DddA protein and prevent cell inactivation. In order to facilitate the purification of DddA protein in subsequent experiments, the corresponding immune protein DddIA was searched within 10kb upstream and downstream of the screened DddA homologous proteins; the screened proteins had different lengths, and only the DddAtox part was needed, so the sequence was aligned again and truncated to obtain 128 truncated sequences in total where the DddA homologous proteins played a role.

[0066] 1.3. IQ-TREE software was used to construct an evolutionary tree of all the mined proteins and other deaminase family protein sequences reported in the literature (Evolution of the deaminase fold and multiple origins of eukaryoticediting and mutagenic nucleic acid deaminases from bacterial toxin systems), and the tree was visualized using ITOL software. From the figure, it can be concluded that the 128 novel deaminases mined by the established bioinformatics pipeline were classified into the evolutionary branch of SCP1.201, so these proteins do belong to the SCP1.201 family ( Figure 1 ).

[0067] 1.4. In order to further screen for active new deaminases, the AlphaFold 2 software was used to predict the structures of all mined proteins, and TM-align structures were compared with known DddA proteins. Finally, 27 new deaminases with structures similar to known proteins were selected for subsequent experimental verification and analysis. The 27 proteins were numbered as deaminases L8, L64, L65, L67, L70, L71, L72, L74, L78, L79, L80, L81, L83, L84, L85, L86, L88, L89, L90, L91, L115, L135, L136, L138, S45, S57, S76 ( Figure 2 ).

[0068] 1.5. The mined deaminase sequence was optimized for corn codons, and the company was commissioned to synthesize the coding genes of the deaminase and downstream immune protein. Taking deaminase L85 as an example, the deaminase L85 and the immune protein coding gene were connected to the pET-duet-1 vector, and the co-expression vector of the deaminase and downstream immune protein was constructed, which was transferred into E. coli TSR2566 prokaryotic induction expression and protein purification. The nucleotide sequence of the deaminase L85 and immune protein coding gene is SEQ ID NO.1.

[0069] The co-expression vector of the deaminase and the downstream immune protein is named as the pETduet1-L85 vector. The structure of the pETduet1-L85 vector is: the fragment between the BamHI and XhoI sites of the pET-duet-1 vector (the small fragment between the BamHI and XhoI sites) is replaced by a DNA molecule whose nucleotide sequence is SEQ ID NO.1, and the other nucleotide sequences of the pET-duet-1 vector are kept unchanged. Among them, the 2nd to 388th positions of SEQ ID NO.1 are the coding gene of the deaminase L85, the 454th to 472nd positions are the T7 promoter, and the 540th to 920th positions are the coding gene of the immune protein.

[0070] The pETduet1-L85 vector can express L85 fusion protein and L85 immune protein with vector sequence and His tag, the amino acid sequence of the L85 fusion protein is SEQ ID NO.2, SEQ ID NO.2 positions 1-10 are the amino acid sequence with His tag encoded by the start codon on the vector to the sequence, positions 11-14 are linker, and positions 15-143 are the amino acid sequence of deaminase L85. The amino acid sequence of the L85 immune protein is SEQ ID NO.3.

[0071] Schematic diagram of deaminase purification system Figure 3 As shown. The steps of inducing expression and protein purification are: transferring the constructed pETduet1-L85 vector into the competent E. coli TSR2566 to obtain the recombinant bacteria TSR2566 / pETduet1-L85. Shake the bacteria in LB liquid medium until OD600=0.6-0.7, add IPTG to induce protein expression, and collect the bacterial solution. The deaminase L85 fusion protein (amino acid sequence is SEQ ID NO.2) obtained by induced expression in the bacterial solution is combined with the immune protein (amino acid sequence is SEQ ID NO.3) to form a protein complex. Then use a nickel column to purify the bacterial solution to obtain a protein complex of deaminase L85 and immune protein. Then use urea solutions of different concentrations of 8M, 6M, 4M, 2M, and 1M to denature and renature the protein complex, and finally the eluate containing only deaminase L85 is purified by molecular sieve to obtain deaminase L85.

[0072] The preparation of the other 26 deaminases was based on deaminase L85, the only difference being the coding sequence of the deaminases.

[0073] Taking deaminase DddA as a control, the preparation method of deaminase DddA refers to deaminase L85, the only difference being that the DNA molecule of SEQ ID NO.1 in the pETduet1-L85 vector is replaced with the DNA molecule shown in SEQ ID NO.4. Among them, positions 2-415 of SEQ ID NO.4 are the coding gene of deaminase DddA, positions 484-502 are T7 promoters, and positions 570-1001 are the coding gene of immune protein of deaminase DddA. The deaminase DddA (amino acid sequence is SEQ ID NO.5) obtained by induced expression is complexed with immune protein (amino acid sequence is SEQ ID NO.6) to form a protein complex.

[0074] Example 2: In vitro activity verification

[0075] 2.1. In vitro deamination activity

[0076] The DNA substrate is labeled with 5'-FAM fluorophore. The deamination reaction system is prepared with 20nM concentration of deaminase to analyze the deamination activity. If the deaminase has deamination activity, the DNA substrate will be cut from the deaminated cytosine. The reaction product of the deamination reaction system has a cleavage band near 18nt ( Figure 4 ).

[0077] The deamination reaction system is as follows: 50 μL L85 protein (20 nM) and 10 μL deamination buffer solution containing DNA substrate are mixed to obtain a reaction mixture. The deamination buffer solution containing DNA substrate is composed of: 20 m M MES, 200 mM NaCl, 1 mM DTT, 80 g / L Ficoll 70, 1 μM DNA substrate.

[0078] The deamination reaction conditions are as follows: subject the reaction mixture to deamination reaction at 37°C for 1 hour; then, 3μL UDG (Uracil-DNA Glycosylase) and 7μL 10×UDG buffer are added to the system, and the enzyme digestion reaction is carried out at 37°C for 30 minutes; 7μL 1M NaOH is added to the system, and the reaction is terminated at 95°C for 3 minutes. The deamination reaction samples obtained are used for subsequent electrophoresis verification.

[0079] Electrophoresis verification: Before electrophoresis, the marker system was: 1 μL of 36nt substrate, 1 μL of 18nt substrate, 5 μL of 2× RNA loading buffer, 13 μL of ddH2O; 77 μL of deamination reaction sample was mixed with 10 μL of 2× RNA loading buffer and placed at 100°C for denaturation for 5 min. During gel electrophoresis, the voltage was adjusted to 150 V for 44 min after working at 100 V for 30 min.

[0080] Result analysis: DNA fragments labeled with 5'-FAM fluorophore can show bands when scanned with a gel scanner (model: iBright FL1500 imaging system). DNA fragments without 5'-FAM fluorophore labeling do not show bands when scanned with a gel scanner. That is, if the deaminase has in vitro deamination activity, the fluorescently labeled DNA substrate will be deaminated and denatured, and the product will contain a fluorescently labeled 18nt DNA single strand. The reaction product of the deamination reaction system will show a band near 18nt when scanned with a gel scanner; if the deaminase has no in vitro deamination activity (or the activity is very weak), the reaction product of the deamination reaction system will show no band near 18nt when scanned with a gel scanner.

[0081] The deamination activity of the above 28 candidate proteins was verified, and a total of 18 deaminases with deamination activity were successfully verified (L8, L70, L71, L72, L78, L79, L80, L91, L115, L138, S57, L83, L84, L85, L86, L89, L90, S45). The verification was repeated again, and based on the results of the two verifications, five deaminases with higher deamination activity, L8, L70, L85, L91, L90, and S45, were finally selected for subsequent verification ( Figure 5 ).

[0082] 2.2 Verification of target sequence preference

[0083] The deamination reaction system was prepared according to 2.1 and the deamination reaction was carried out. The difference from 2.2 was that the DNA substrate was different. N in the DNA substrate represented four different bases: ATGC. Figure 6 ) to determine the preference of the candidate deaminase for the target sequence C in vitro. The results showed that deaminase L85 can effectively deaminize cytosine in vitro in the context of NC (N is any base) Figure 7 ).

[0084] Table 1 Sequences in this application

[0085]

[0086]

[0087]

[0088] The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experimental conditions, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In a word, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including departure from the disclosed scope in the present application and changes made with conventional techniques known in the art.

Claims

1. Protein, characterized in that: The protein is any of the following: A1) a protein whose amino acid sequence is SEQ ID NO. 2; A2) a protein whose amino acid sequence is positions 15 to 143 of SEQ ID NO. 2; A3) a protein related to deaminase having an amino acid sequence represented by A1) or A2) with substitution and / or deletion and / or addition of amino acid residues, having 80% or more identity with the protein represented by A1) or A2); A4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1), A2) or A3).

2. A biological material related to the protein according to claim 1, wherein the biological material is any one of the following: B1) a nucleic acid molecule encoding the protein; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) a transgenic plant cell line containing the nucleic acid molecule described in B1), a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3); B6) transgenic plant tissue containing the nucleic acid molecule described in B1), transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3); B7) A transgenic plant organ containing the nucleic acid molecule described in B1), a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3).

3. The biomaterial according to claim 2, characterized in that: B1) The nucleic acid molecule is at least one of the following: g1) the coding sequence of the coding strand is a cDNA molecule or a DNA molecule shown in positions 2 to 388 of SEQ ID NO.1; g2) A cDNA molecule or a DNA molecule that has 80% or more identity with the nucleotide sequence defined in g1) and encodes the protein.

4. Use of the protein according to claim 1 as cytidine deaminase.

5. Use of the protein according to claim 1 in the preparation of cytidine deaminase.

6. Use of the biomaterial according to claim 2 or 3 in the preparation of cytidine deaminase.

7. Use of the protein according to claim 1 in single base editing.

8. Use of the protein according to claim 1 in the preparation of single-base editing products.

9. Use of the biomaterial described in claim 2 or 3 in single base editing.

10. Use of the biomaterial according to claim 2 or 3 in the preparation of single-base editing products.

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