Single-base editor, deaminase used by single-base editor and application of deaminase

Abstract

The invention discloses a single-base editor, deaminase used by the single-base editor and application of the single-base editor, and belongs to the technical field of genetic engineering. The technical problems to be solved in the invention are as follows: natural cytosine deaminase without sequence preference is mined, a base editor is constructed, and the base editing efficiency and range are improved. In order to solve the technical problem, the application provides a cytosine base editor, the cytosine base editor is a fusion protein, the fusion protein is a protein containing cytidine deaminase, a Cas protein and a uracil glycosylase inhibitor, and the cytidine deaminase is a protein at the 28th-164th site of an amino acid sequence SEQ ID NO.2. The invention also provides application of the fusion protein and related biological materials thereof in plant single base editing. The single-base editor provided by the invention can improve the efficiency of cytosine base editing, can accurately mediate the base mutation of a target site, and can be widely applied to corn and even other plant cells.

Description

Technical Field

[0001] The present application belongs to the field of genetic engineering technology, and specifically relates to a single-base editor and the deaminase used therein and its application. 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, and generating sequence insertions, deletions, and substitutions. Currently, commonly used genome editing technology platforms include: ZFN, TALEN, and CRISPR / Cas systems. Among them, the CRISPR / Cas genome editing technology platform is the most 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 technical problem to be solved by this application is to explore the natural cytosine deaminase with no sequence preference, construct a base editor, and improve the efficiency and scope of base editing.

[0006] In order to solve the above-mentioned technical problems, the present application provides a cytosine base editor, which is a fusion protein.

[0007] The fusion protein is a protein containing cytidine deaminase (cytosine deaminase), Cas protein and uracil glycosylase inhibitor, the cytidine deaminase is protein L8, and the protein L8 is any one of the following,

[0008] A1) a protein whose amino acid sequence is positions 28 to 164 of SEQ ID NO. 2;

[0009] A2) a protein related to deaminase obtained by substituting, deleting and / or adding amino acid residues of the amino acid sequence shown in A1);

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

[0011] Furthermore, the connection described in A3) can be via a peptide bond.

[0012] 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.

[0013] Furthermore, in the fusion protein, the Cas protein may be nCas9.

[0014] Furthermore, in the fusion protein, the nCas9 may be a protein whose amino acid sequence is positions 182-1548 of SEQ ID NO.2.

[0015] Furthermore, in the fusion protein, the uracil glycosylase inhibitor may be a protein having an amino acid sequence of positions 1559-1641 of SEQ ID NO.2 and / or positions 1652-1734 of SEQ ID NO.2.

[0016] Furthermore, in the fusion protein, the amino acid sequence of the nuclear localization signal may be SEQ ID NO.2, No. 3-9 and / or SEQ ID NO.2, No. 1766-1781.

[0017] Furthermore, the fusion protein may be a protein whose amino acid sequence is SEQ ID NO.2.

[0018] The present application also provides a biological material related to the above-mentioned fusion protein, and the biological material may be at least one of the following D1)-D7):

[0019] D1) a nucleic acid molecule encoding the fusion protein;

[0020] D2) an expression cassette containing the nucleic acid molecule described in D1);

[0021] D3) a recombinant vector containing the nucleic acid molecule described in D1) or a recombinant vector containing the expression cassette described in D2);

[0022] D4) a recombinant microorganism containing the nucleic acid molecule described in D1), a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3);

[0023] D5) a transgenic plant cell line containing the nucleic acid molecule described in D1), a transgenic plant cell line containing the expression cassette described in D2), or a transgenic plant cell line containing the recombinant vector described in D3);

[0024] D6) transgenic plant tissue containing the nucleic acid molecule described in D1), transgenic plant tissue containing the expression cassette described in D2), or transgenic plant tissue containing the recombinant vector described in D3);

[0025] D7) A transgenic plant organ containing the nucleic acid molecule described in D1), a transgenic plant organ containing the expression cassette described in D2), or a transgenic plant organ containing the recombinant vector described in D3).

[0026] Furthermore, in the biological material, the DNA molecule in D1) may be at least one of the following:

[0027] D11) the nucleotide sequence of the coding strand is the cDNA molecule or DNA molecule shown in positions 3589-8899 of SEQ ID NO.1;

[0028] D12) the nucleotide sequence of its coding strand is the cDNA molecule or DNA molecule shown in SEQ ID NO.1, positions 3514-9040;

[0029] D12) is a cDNA molecule or a DNA molecule that has 80% or more identity with the nucleotide sequence defined in D11) and / or D12) and encodes the fusion protein.

[0030] In order to solve the above technical problems, the present application also provides the above protein L8 and related biological materials.

[0031] The protein L8 is any of the following:

[0032] A1) a protein whose amino acid sequence is positions 28 to 164 of SEQ ID NO. 2;

[0033] The amino acid sequences shown in A2) and A1) are substituted and / or deleted and / or added to obtain a protein that has more than 80% identity with the protein shown in A1) and is related to deaminase;

[0034] A3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0035] Furthermore, the connection described in A3) can be via a peptide bond.

[0036] 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.

[0037] In some embodiments of the present application, the tag is a His tag.

[0038] The present application also provides a biological material related to the above-mentioned protein L8, wherein the biological material may be at least one of the following:

[0039] B1) a nucleic acid molecule encoding the protein;

[0040] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0041] B3) a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2);

[0042] 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);

[0043] 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);

[0044] 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);

[0045] 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).

[0046] Furthermore, in the biological material, the nucleic acid molecule in B1) may be at least one of the following:

[0047] B11) the coding sequence of the coding strand is the cDNA molecule or DNA molecule shown in positions 2 to 412 of SEQ ID NO.4;

[0048] B12) the nucleotide sequence of the coding strand is the cDNA molecule or DNA molecule shown in positions 3589-4189 of SEQ ID NO.1;

[0049] B13) has 80% or more identity with the nucleotide sequence defined in B11) and / or B11), and encodes a cDNA molecule or a DNA molecule of the protein L8.

[0050] In the present application, the expression cassette refers to a DNA capable of expressing the protein in a host cell (such as a plant 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 1 0099169.7)), 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).

[0051] In some embodiments of the present application, the expression cassette uses the Zmubi promoter (nucleotide sequence is SEQ ID NO.1 1506-3498) to start the expression of deaminase L8, DddA, linker, nCas9 (D10A) and glycosylase inhibitor protein (UGI) fusion protein, and the expression is terminated by the RBCS E9T terminator (nucleotide sequence is SEQ ID NO.1 9044-9678). The expression cassette is connected by the Zmubi promoter, the nucleic acid molecule encoding the cytosine base editor and the RBCS E9T terminator.

[0052] 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.

[0053] 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.

[0054] In the above, the recombinant microorganism can specifically be bacteria, yeast, algae and fungi.

[0055] 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.

[0056] The present application also provides the use of the above-mentioned protein L8, biological materials related to protein L8, the fusion protein and / or biological materials related to the fusion protein in single-base editing and / or preparation of single-base editing products.

[0057] Furthermore, in the present application, the single-base editing can occur outside the cell or inside the cell.

[0058] Furthermore, in the present application, the single base editing can occur in plant cells.

[0059] Furthermore, in the present application, the single-base editing can occur within the organelles of plant cells.

[0060] The present application also provides a method for mutating C:G on a plant genome to T:A.

[0061] The method provided in the present application for mutating the C:G base pair on the plant genome to T:A comprises the following steps: introducing a DNA molecule expressing the cytosine base editor and sgRNA into a recipient plant to obtain a target plant in which C:G is mutated to T:A; the target sequence of the sgRNA is 5′-N19-20PAM-3′, and the N19-20 is 19-20 Ns.

[0062] In the above method, the Cas protein in the cytosine base editor may be nCas9, the PAM (protospacer adjacent motif) is NGG; and the N is A, G, C or T.

[0063] When the DNA molecule expressing the cytosine base editor and sgRNA is introduced into the recipient plant, the PEG-mediated transformation method can be used, or the gene editing tool kit can be introduced into the corn protoplast or callus by using one of the gene gun method or Agrobacterium infection method, which is easily understood by those skilled in the art. It is well known to those skilled in the art that the corn genomic DNA consists of two chains, so the target nucleotide sequence can be on any complementary chain. For example, when the target nucleotide sequence is located in the positive chain of a functional gene, if the C at a specific site of the functional gene is site-directedly mutated to T and / or G is site-directedly mutated to A, and if one of the mutations can obtain the expected amino acid in the corresponding functional protein, this system can also be used to achieve the goal, that is, the C in the triplet codon can be replaced by T and / or G can be replaced by A by directly performing base replacement on the positive chain, thereby obtaining a corn gene function "corrected" mutant; or when the target nucleotide sequence is located in the antisense chain of a functional gene, if the C at a specific site of the functional gene is site-directedly mutated to T, and if one of the mutations can obtain the expected amino acid in the corresponding functional protein, this system can be used to achieve the goal, that is, the amino acid encoded by the triplet codon in the positive chain can be changed by site-directedly mutating G in the antisense chain to A, thereby replacing the corresponding complementary C in the positive chain with T, thereby obtaining a corn gene function "corrected" mutant.

[0064] In the above, the plant may be a dicotyledon or a monocotyledon. The monocotyledon may be corn. The single base editing may be replacing cytosine C with uracil.

[0065] The technical effects achieved by this application are as follows:

[0066] This application uses metagenomic big data to conduct a comprehensive and systematic bioinformatics mining analysis on the new cytidine deaminase, and selects a protein L8 that is similar to the known DddA protein structure but has a low sequence similarity (identity <63.04%) for functional verification, and uses it to develop a new CRISPR / Cas gene editing toolbox. The editing efficiency of the cytosine base editing technology mediated by the fusion of the deaminase L8 and nCas9 (D10A) in this application is significantly higher than that of the DddA-mediated cytosine base editing technology, and its editing activity window can be expanded, thereby improving the efficiency of the cytosine base editing technology and broadening its scope of application. Provide a set of important gene function research and correction tools for researchers in the field of plant research and crop genetic improvement. The single-base editor of this application can improve the efficiency of cytosine base editing and can accurately mediate base mutations at the target site, and can be widely used in corn and even other plant cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 It is an analysis system for the in vitro deamination activity of deaminase.

[0068] Figure 2 is the in vitro deamination activity of deaminase L8.

[0069] Figure 3 To verify the DNA substrate structure in the target sequence preference system in vitro.

[0070] Figure 4 These are the results of validating the target sequence preference of deaminase L8 for in vitro deamination.

[0071] Figure 5 The position and structure of the target fragment in the L8 vector.

[0072] Figure 6 Fluorescence detection of maize protoplasts L8 and DddA.

[0073] Figure 7 It is a single base editing vector for the deaminase L8 and DddA systems.

[0074] Figure 8 The editing window and editing efficiency of deaminase L8 and DddA on the Bx9 gene are demonstrated.

[0075] Fig. 9 Demonstration of sequence preference for editing of deaminase L8 and DddA on the Bx9 gene. DETAILED DESCRIPTION

[0076] 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.

[0077] 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.

[0078] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0079] The following examples used GraphPad Prism statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. Two-way ANOVA was used, with * representing P < 0.05, ** representing P < 0.01, *** representing P < 0.001, and **** representing P < 0.0001.

[0080] Example 1: Novel cytidine deaminase mining process

[0081] 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.

[0082] 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.

[0083] 1.3、Using IQ-TREE software, we constructed an evolutionary tree with 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 visualized it using ITOL software. From the figure, we can conclude 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.

[0084] 1.4. In order to further screen out active new deaminases, 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 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, and S76.

[0085] 1.5. The mined deaminase sequence was optimized for corn codons, and the company was commissioned to synthesize the coding genes of deaminase and downstream immune protein. Taking deaminase L8 as an example, the deaminase L8 and immune protein coding genes were connected to the pET-duet-1 vector, and the co-expression vector of deaminase and downstream immune protein was constructed, which was transferred into E. coli TSR2566 prokaryotic induction expression and protein purification. The coding genes of deaminase L8 and immune protein are DNA molecules with the nucleotide sequence of SEQ ID NO.4.

[0086] The co-expression vector of deaminase and downstream immune protein is named pETduet1-L8 vector. The structure of pETduet1-L8 vector is: replace the fragment between BamHI and XhoI sites of pET-duet-1 vector (small fragment between BamHI and XhoI sites) with a DNA molecule whose nucleotide sequence is SEQ ID NO.4, and keep the other nucleotide sequences of pET-duet-1 vector unchanged. Among them, SEQ ID NO.4 No. 2-412 is the coding gene of deaminase L8, No. 478-496 is the T7 promoter, and No. 564-944 is the coding gene of immune protein.

[0087] The pETduet1-L8 vector can express L8 fusion protein with a vector sequence and a His tag and an immune protein of L8 (amino acid sequence is shown in Table 2). The amino acid sequence of the L8 fusion protein is from the start codon on the vector to the amino acid sequence with a His tag encoded by the sequence, positions 11-14 are linkers, and positions 15-151 are the amino acid sequence of the deaminase L8.

[0088] The steps of inducing expression and protein purification are as follows: the constructed pETduet1-L8 vector is transferred into the competent E. coli TSR2566 to obtain the recombinant bacteria TSR2566 / pETduet1-L8. The bacteria are shaken in LB liquid medium to OD600 = 0.6-0.7, IPTG is added to induce protein expression, and the bacterial solution is collected. Then the bacterial solution is purified using a nickel column to obtain the protein complex of the L8 fusion protein (amino acid sequence is shown in Table 2) and the L8 immune protein (amino acid sequence is shown in Table 2). Then, urea solutions of different concentrations of 8M, 6M, 4M, 2M, and 1M are used to denature and renature the protein complex, and finally the eluate containing only the deaminase L8 is purified by molecular sieve to obtain the L8 fusion protein.

[0089] The preparation of the other 26 deaminases was based on deaminase L8, the only difference being the coding sequence of the deaminases.

[0090] With deaminase DddA as a control, the preparation method of deaminase DddA refers to deaminase L8, the only difference being that the DNA molecule of SEQ ID NO.4 in the pETduet1-L8 vector is replaced with the DNA molecule shown in SEQ ID NO.5. Positions 2-415 of SEQ ID NO.5 are the coding gene of deaminase DddA, positions 484-502 are T7 promoters, and positions 570-1001 are the coding gene of the immune protein of deaminase DddA. The deaminase DddA fusion protein (amino acid sequence shown in Table 2) obtained by induced expression is complexed with the immune protein of DddA (amino acid sequence shown in Table 2) to form a protein complex.

[0091] Example 2: In vitro activity verification

[0092] 2.1. In vitro deamination activity

[0093] 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 1 ).

[0094] The deamination reaction system is as follows: 50 μL L8 protein (20 nM) and 10 μL deamination buffer solution containing DNA substrate are mixed to obtain a deamination 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.

[0095] The deamination reaction conditions are as follows: subject the above deamination reaction mixture to deamination reaction at 37°C for 1 hour; then, 3 μL UDG (Uracil-DNA Glycosylase) and 7 μL 10×UDG buffer are added to the system, and the enzyme digestion reaction is carried out at 37°C for 30 minutes; 7 μL 1M NaOH is added to the system, and the reaction is terminated at 95°C for 3 minutes, and the obtained deamination reaction sample is used for subsequent electrophoresis verification.

[0096] 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.

[0097] 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.

[0098] 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. Among them, the deamination activity verification results of deaminase L8 are shown in Figure 2 .

[0099] 2.2 Verification of target sequence preference

[0100] 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 3) to determine the preference of candidate deaminases for the target sequence C in vitro. The results showed that deaminase L8 can effectively deaminize cytosine in the context of NC (N is any base) Figure 4 ). Among them, deaminase L8 showed better activity. Therefore, deaminase L8 was used for subsequent experimental verification.

[0101] Example 3: Functional verification of deaminase L8 in eukaryotes

[0102] 3.1. Expression verification of deaminase L8 in eukaryotic cells

[0103] Since the discovered deaminase L8 is a double-stranded cytidine deaminase, it does not require DNA chain breaks during the deamination process. Therefore, there is also a problem that cannot be ignored, that is, L8 is cytotoxic, which will cause vector mutations or directly cause cell death during the vector construction process. In order to solve this problem, a cat1 intron sequence was inserted between the L8 gene and the DddA gene, thereby ensuring that the L8 and DddA genes are not expressed during the vector construction, so that the vector construction can be carried out smoothly.

[0104] In order to verify that the intron sequences inserted into L8 and DddA can be correctly cleaved in maize LH244 protoplasts, a vector was constructed to drive transcription and expression of L8 and eGFP proteins with introns using a 35s promoter ( Figure 5 ), named L8 vector. L8 vector was used to transform corn protoplasts, and the positive corn protoplasts obtained by transformation were represented by L8. The protoplast transformation steps refer to the literature "Targeted large fragment deletion in plants using paired crRNAs with type I CRISPR system".

[0105] A vector expressing DddA and eGFP proteins with intron was constructed by 35s promoter, named DddA vector. Maize protoplasts were transformed with pUC-eGFP vector and DddA vector as control. The positive maize protoplasts obtained by transformation were represented by pUC-eGFP and DddA, respectively.

[0106] Among them, the L8 vector structure is: replace the fragment between the BamHI and XhoI restriction enzyme recognition sites of pUC-eGFP with a DNA molecule having a nucleotide sequence of SEQ ID NO.6, and keep the other nucleotide sequences of the backbone vector unchanged. SEQ ID NO.6 No. 4-21 is the nucleotide sequence of the 6×His tag, No. 22-33 is the nucleotide sequence of the linker, No. 34-334 is the deaminase L8 N-terminal encoding gene, No. 335-524 is the nucleotide sequence of cat1 intron, and No. 525-634 is the deaminase L8 C-terminal encoding gene. The nucleotide sequence of the pUC-eGFP vector is shown in Table 2, wherein the nucleotide sequence of the pUC-eGFP vector No. 1137-1142 is the BamHI restriction enzyme recognition site, No. 1161-1166 is the XhoI restriction enzyme recognition site, No. 744-1089 is the 35s promoter, and No. 1182-1901 is eGFP.

[0107] The structure of the DddA vector is different from that of the L8 vector only in that the DNA molecule whose nucleotide sequence is SEQ ID NO.6, positions 34-634 (deaminase L8 N-terminal encoding gene, cat1 intron and deaminase L8 C-terminal encoding gene) in the L8 vector is replaced with a DNA molecule whose nucleotide sequence is SEQ ID NO.3. Among them, positions 1-217 of SEQ ID NO.3 are the nucleotide sequence of the deaminase DddA N-terminal encoding gene, positions 218-407 are the nucleotide sequence of the cat1 intron, and positions 408-604 are the nucleotide sequence of the deaminase DddA C-terminal encoding gene.

[0108] If the intron sequence can be cut correctly, the GFP protein can be expressed and can emit light under a fluorescence microscope. Otherwise, GFP will be frameshifted and no green fluorescence can be seen under a fluorescence microscope. The results show that maize protoplasts L8, DddA and pUC-eGFP can be observed to emit green fluorescence under a microscope ( Figure 6 ), indicating that the introns in the L8 gene and DddA gene can be cleaved in maize protoplasts.

[0109] 3.2 Construction of single-base editing vector

[0110] Subsequently, single-base editing vectors of the deaminase L8 and DddA systems were designed, and deaminase DddA was used as a control to verify whether deaminase L8 had deamination activity in plants. The Osu3 promoter was used to initiate the expression of sgRNA; the Zmubi promoter was used to initiate the expression of deaminase L8 or DddA, linker, nCas9 (D10A) and glycosylase inhibitor protein (UGI) fusion proteins. They were integrated into the pUC19 vector (NEB product, catalog number N3041S) through homologous recombination, and the obtained recombinant vectors were named pUC19-L8 vector and pUC19-DddA vector, respectively.

[0111] The nucleotide sequence of the pUC19-L8 vector is SEQ ID NO. 1. pUC19-L8 can express a fusion protein having an amino acid sequence of SEQ ID NO. 2.

[0112] SEQ ID NO.1, positions 57-1493 are the nucleotide sequence of the sgRNA gene expression cassette. Among them: SEQ ID NO.1, positions 57-436 are the nucleotide sequence of the OsU3 promoter; SEQ ID NO.1, positions 438-444 and positions 1120-1127 are the BsaI restriction enzyme recognition sites; SEQ ID NO.1, positions 1127-1202 are the sgRNA scaffold; SEQ ID NO.1, positions 1203-1493 are the nucleotide sequence of the OsU3 terminator.

[0113] Positions 3508-9043 of SEQ ID NO.1 are the nucleotide sequence of the expression cassette of the deaminase L8, nCas9 (D10A) and glycosylase inhibitor protein (UGI) fusion protein. Wherein: SEQ ID NO.1 positions 1506-3498 are the nucleotide sequence of Zmubi promoter; SEQ ID NO.1 positions 3514-3534 are the nucleotide sequence of SV40 NLS, positions 3535-3558 are the nucleotide sequence of linker1, positions 3559-3576 are the nucleotide sequence of 6×His tag, positions 3577-3588 are the nucleotide sequence of linker2, positions 3589-3889 are the nucleotide sequence of deaminase L8 N-terminal encoding gene, positions 3890-4079 are the nucleotide sequence of cat1intron, positions 4080-4189 are the nucleotide sequence of deaminase L8 C-terminal encoding gene, positions 4190-4240 are the nucleotide sequence of A(EAAAK)3A linker, positions 4241-8341 are the nucleotide sequence of nCas9 encoding gene, and positions 8342-8371 are 10aa The nucleotide sequence of the linker is shown in Figure 1. The nucleotide sequence of the UGI coding gene is shown in Figure 1. The nucleotide sequence of the UGI coding gene is shown in Figure 1. The nucleotide sequence of the UGI coding gene is shown in Figure 1. The nucleotide sequence of the UGI coding gene is shown in Figure 1. The nucleotide sequence of the UGI coding gene is shown in Figure 1. The nucleotide sequence of the UGI coding gene is shown in Figure 1. The nucleotide sequence of the UGI coding gene is shown in Figure 1. The nucleotide sequence of the UGI coding gene is shown in Figure 1. The nucleotide sequence of the UGI coding gene is shown in Figure 1. The nucleotide sequence of the UGI coding gene is shown in Figure 1. The nucleotide sequence of the UGI coding gene is shown in Figure 1. The nucleotide sequence of the UGI coding gene is shown in Figure 1. The nucleotide sequence of the UGI coding gene is shown in Figure 1. The nucleotide sequence of the UGI coding gene is shown in Figure 1. The nucleotide sequence of the UGI coding gene is shown in Figure 1.

[0114] SEQ ID NO.2: 3-9 is the amino acid sequence of SV40 NLS, 10-17 is the amino acid sequence of linke1, 18-23 is the amino acid sequence of 6×His tag, 24-27 is the amino acid sequence of linker2, 28-164 is the amino acid sequence of deaminase L8, 165-181 is the amino acid sequence of A(EAAAK)3A linker, 182-1548 is the amino acid sequence of nCas9, 1549-1558 is the amino acid sequence of 10aa linker, 1559-1641 is the amino acid sequence of UGI, 1642-1651 is the amino acid sequence of 10aa linker, 1652-1734 is the amino acid sequence of UGI, 1735-1738 is the amino acid sequence of 4aa The amino acid sequence of the linker is as follows: the amino acid sequence of 1739-1765 is that of 3×HA (81), and the amino acid sequence of 1766-1781 is that of NLS.

[0115] The structure of the pUC19-DddA vector is the same as that of the pUC19-L8 vector, except that the DNA molecule whose nucleotide sequence is SEQ ID NO.1 at positions 3589-4189 in the pUC19-L8 vector is replaced with a DNA molecule whose nucleotide sequence is SEQ ID NO.3. The pUC19-DddA vector expresses a base editor containing the DddA protein (a fusion protein containing the DddA protein), and the amino acid sequence of the base editor containing the DddA protein is shown in Table 2.

[0116] 3.3. In order to detect the single-base editing activity of L8, the target site of NGG (SEQ ID NO.7: CTGGCCCGCACCGTCACAG) was designed on the maize Bx9 (Zm00001eb033030) gene, and the annealing primer was designed to anneal and synthesize a DNA fragment with a sticky end for standby use. The pUC19-L8 vector was digested with BsaI to obtain the digestion product, and the obtained DNA fragment and the digestion product were connected with T4 enzyme to obtain the recombinant vector pUC19-L8-Bx9 vector targeting the Bx9 gene. The structure of the pUC19-L8-Bx9 vector is: the fragment between the BsaI digestion recognition sites of the pUC19-L8 vector (the small fragment between the BsaI digestion recognition sites) is replaced by a DNA molecule with a nucleotide sequence of SEQ ID NO.7, and the other nucleotide sequences of the pUC19-L8 vector remain unchanged. The pUC19-L8-Bx9 vector was extracted into a transfection-grade plasmid and then transformed into corn protoplasts. After 48 h of culture, the DNA of the corn transformed plasmids was extracted. Targets were designed upstream and downstream of the target, and a fragment of about 250 bp was amplified to construct a DNA library for next-generation sequencing.

[0117] The nucleotide sequences of the annealing primers are as follows:

[0118] Bx9T-F: ATATATGGTCTCTGGCG CTGGCCCGCACCGTCACAG GTT;

[0119] Bx9T-R:ATTATTGGTCTCTAAAC CTGTGACGGTGCGGGCCAG C.

[0120] The cytosine base editor constructed by deaminase DddA was used as a control. The preparation method of the pUC19-DddA-Bx9 vector refers to the pUC19-L8-Bx9 vector, with the only difference being that the pUC19-L8 vector was replaced by the pUC19-DddA vector.

[0121] After analyzing the results of the second-generation sequencing, the ratio of C to T and G to A within the target range was counted, and the N of PAM NGG was defined as 0, the upstream was recorded as a negative value, and the downstream was recorded as a positive value. The results showed that on the Bx9 gene, the editing window range of deaminase L8 and DddA was about 140bp; the editing efficiency of deaminase L8 at most sites was significantly higher than that of deaminase DddA, especially at G-74, L8 had the highest editing efficiency of 19.25%, and the average was 18.2% ( Figure 8 ).

[0122] In summary, it can be proved that the excavated L8 is active and shows higher editing activity than DddA at multiple sites of the Bx9 target gene.

[0123] Table 1 Statistical results of editing efficiency of L8 and DddA

[0124]

[0125] Note: CK refers to protoplast cells that have not undergone any treatment.

[0126] 3.4. In order to detect the sequence preference of L8 and DddA in Bx9 gene editing, 3 bp upstream and downstream of the effective editing site were selected, and the base distribution was statistically analyzed using weblogo software after sequence alignment. The results showed that DddA has a sequence preference for TC sequence in Bx9 gene as described by previous researchers. L8 can target HC (H is A, C or T) deamination ( Fig. 9 ).

[0127] Table 2 Partial sequences in this application

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134] 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. A fusion protein, characterized in that: The fusion protein is a protein containing cytidine deaminase, Cas protein and uracil glycosylase inhibitor, the cytidine deaminase is protein L8, and the protein L8 is shown in any one of the following A1)-A3): A1) a protein whose amino acid sequence is positions 28 to 164 of SEQ ID NO. 2; A2) a protein related to deaminase obtained by substituting, deleting and / or adding amino acid residues of the amino acid sequence shown in A1); A3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

2. The fusion protein according to claim 1, characterized in that: The Cas protein is nCas9.

3. The fusion protein according to claim 1 or 2, characterized in that: The fusion protein is a protein formed by connecting the cytidine deaminase, the Cas protein, the uracil glycosylase inhibitor and the nuclear localization signal.

4. The fusion protein according to claim 3, characterized in that: The fusion protein is a protein whose amino acid sequence is SEQ ID NO.

2.

5. The biological material related to the fusion protein according to any one of claims 1 to 4 is at least one of the following D1) to D7): D1) a nucleic acid molecule encoding the fusion protein; D2) an expression cassette containing the nucleic acid molecule described in D1); D3) a recombinant vector containing the nucleic acid molecule described in D1) or a recombinant vector containing the expression cassette described in D2); D4) a recombinant microorganism containing the nucleic acid molecule described in D1), a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3); D5) a transgenic plant cell line containing the nucleic acid molecule described in D1), a transgenic plant cell line containing the expression cassette described in D2), or a transgenic plant cell line containing the recombinant vector described in D3); D6) transgenic plant tissue containing the nucleic acid molecule described in D1), transgenic plant tissue containing the expression cassette described in D2), or transgenic plant tissue containing the recombinant vector described in D3); D7) A transgenic plant organ containing the nucleic acid molecule described in D1), a transgenic plant organ containing the expression cassette described in D2), or a transgenic plant organ containing the recombinant vector described in D3).

6. The biomaterial according to claim 5, characterized in that: D1) The DNA molecule is at least one of the following: D11) the nucleotide sequence of the coding strand is the cDNA molecule or DNA molecule shown in positions 3589-8899 of SEQ ID NO.1; D12) the nucleotide sequence of its coding strand is the cDNA molecule or DNA molecule shown in SEQ ID NO.1, positions 3514-9040; D12) is a cDNA molecule or a DNA molecule that has 80% or more identity with the nucleotide sequence defined in D11) and / or D12) and encodes the fusion protein.

7. Protein, characterized in that: The protein is the protein L8 described in claim 1.

8. A biological material related to the protein of claim 7, wherein the biological material is at least 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).

9. The biomaterial according to claim 8, characterized in that: B1) The nucleic acid molecule is at least one of the following: B11) the coding sequence of the coding strand is the cDNA molecule or DNA molecule shown in positions 2 to 412 of SEQ ID NO.4; B12) the nucleotide sequence of the coding strand is the cDNA molecule or DNA molecule shown in positions 3589-4189 of SEQ ID NO.1; B13) is a cDNA molecule or a DNA molecule that has 80% or more identity with the nucleotide sequence defined in B11) and / or B11), and encodes the protein.

10. Use of the fusion protein of any one of claims 1-4, the biomaterial of claim 5 or 6, the protein of claim 7 and / or the biomaterial of claim 8 or 9 in single-base editing and / or preparation of single-base editing products.

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