Enhanced cytosine deaminase, base editing system comprising same and application

Abstract

This application relates to the field of gene editing technology, specifically to an enhanced cytosine deaminase, a base editing system containing it, and its applications. This application extensively collected and integrated datasets from published literature, which contain sequence information and related biological characteristics of multiple known enzymes. Based on these datasets, cytosine deaminases from various sources were screened and their sequences were compared with functional sequences. Combined with structural prediction and functional verification, a cytosine deaminase (ch-YZ88-CBE) derived from *Actinomyces biskii*, suitable for chicken cells and capable of efficient C-to-T editing, was successfully screened. A CBE system with good editing activity and stability was constructed, providing key technical support for avian genetic improvement and functional gene research.

Description

Technical Field

[0001] This application relates to the field of gene editing technology, specifically to an enhanced cytosine deaminase, a base editing system containing the same, and its applications. Background Technology

[0002] While CRISPR technology has flourished, a series of more advanced and sophisticated gene-editing technologies have emerged. Base editing technology can precisely replace individual bases without disrupting the overall double helix structure of DNA, offering new hope and strategies for tackling many genetic diseases caused by single-base mutations. Guided editing technology enables the precise insertion, deletion, or replacement of small DNA segments, providing more flexible and diverse tools for constructing complex disease models and deeply analyzing gene function, helping scientists to better understand the pathogenesis of diseases. Retrotransposon technology can purposefully integrate large segments of DNA into the genome, providing a more robust and efficient vector solution for introducing functional genes and repairing missing genes in gene therapy, injecting new vitality into the development of gene therapy.

[0003] However, despite the remarkable achievements in gene editing technology, a series of problems and challenges remain to be solved in its practical application. Taking base editing technology as an example, existing cytosine deaminases often suffer from low editing efficiency, insufficient specificity, and off-target effects when performing base editing tasks. Off-target effects can lead to unexpected gene editing at non-target sites, causing unpredictable gene changes and potentially negatively impacting cell function and organismal health. Low editing efficiency means that more time, effort, and resources are needed to achieve the desired gene editing effect, which to some extent limits the promotion of gene editing technology in clinical treatment and large-scale applications. Furthermore, the preference of existing cytosine deaminases for specific sequences also makes them ineffective when editing certain specific gene sequences, failing to meet diverse gene editing needs.

[0004] Therefore, given the current lack of efficient and stable base editing tools in chicken-derived systems, it is imperative to conduct targeted research to develop high-quality base editing tools suitable for chicken-derived systems. Summary of the Invention

[0005] This application provides a cytosine deaminase, which has higher editing efficiency, and the specific solution involved is as follows: 1. A cytosine deaminase having the amino acid sequence shown in SEQ ID NO.1.

[0006] 2. The use of the amino acid sequence shown in SEQ ID NO: 1 as a cytosine deaminase.

[0007] 3. The application of the amino acid sequence shown in SEQ ID NO: 1 in the preparation of cytosine deaminase.

[0008] 4. The application of the amino acid sequence shown in SEQ ID NO: 1 as a cytosine deaminase in base editing of the chicken genome.

[0009] 5. A base-editing fusion protein, comprising a cytosine deaminase domain and a nucleic acid targeting domain; The cytosine deaminase domain contains the cytosine deaminase as described in item 1.

[0010] 6. The base-editing fusion protein according to item 5, wherein the nucleic acid targeting domain For TALE, ZFP, or CRISPR effector protein domains; Preferably, the nucleic acid targeting domain is a CRISPR effector protein domain.

[0011] 7. The base-editing fusion protein according to item 6, wherein the CRISPR effector protein is at least one of Cas9, Cpf1, Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, Csn2, Cas4, C2c1(Cas12b), C2c3, C2c2, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12l, Cas12m or other available CRISPR effector proteins; Preferably, the CRISPR effector protein is Cas9, which is nuclease-inactivated Cas9, Cas9 nickase, or Cas9 with nuclease activity.

[0012] 8. The base-editing fusion protein according to any one of items 5 to 7, wherein it further contains a uracil DNA glycosylation inhibitor (UGI).

[0013] 9. The base-editing fusion protein according to any one of claims 5 to 8, wherein the base-editing fusion protein further comprises at least one nuclear localization sequence (NLS).

[0014] 10. A base editing system for modifying the chicken genome, comprising: The cytosine deaminase as described in item 1 or an expression construct containing a nucleotide sequence encoding the cytosine deaminase; or a base-editing fusion protein as described in any one of items 5 to 9; or an expression construct containing a nucleotide sequence encoding the base-editing fusion protein.

[0015] 11. The base editing system according to claim 10, wherein the base editing system further comprises at least one sgRNA and / or at least one expression construct containing a nucleotide sequence encoding said at least one sgRNA; The at least one sgRNA targets at least one site in the chicken genome.

[0016] 12. A method for base editing of a chicken genome, comprising contacting the base editing system of item 10 or 11 with a target site in the chicken genome to achieve base editing of the chicken genome.

[0017] 13. The method according to claim 12, wherein the base editing system contacts the target site to perform deamination, resulting in the substitution of one or more nucleotides in the target site; Preferably, the target site comprises a DNA sequence 5'-MCN-3', where M is A, T, C, or G, and N is A, T, C, or G.

[0018] 14. The method according to item 12 or 13, wherein the contact is performed in vivo or in vitro.

[0019] 15. A method for producing at least one modified chicken cell, comprising: introducing the base editing system of item 10 or 11 into at least one chicken cell, thereby causing substitution of one or more nucleotides within a target site in the at least one chicken cell.

[0020] 16. A polynucleotide encoding a cytosine deaminase as described in item 1 or a base-editing fusion protein as described in any one of items 5 to 9.

[0021] 17. A carrier comprising the polynucleotide as described in item 16.

[0022] 18. A host cell comprising the polynucleotide as described in item 16 and the vector as described in item 17.

[0023] 19. A reagent or kit for base editing of chicken genes, comprising the cytosine deaminase as described in item 1, the base editing fusion protein as described in any one of items 5 to 9, or the base editing system as described in item 10 or 11.

[0024] 20. A composition comprising the cytosine deaminase as described in claim 1, the base editing fusion protein as described in any one of claims 5 to 9, or the base editing system as described in claim 10 or 11.

[0025] 21. The composition according to claim 20, wherein the composition is a pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0026] 22. The use of the cytosine deaminase as described in item 1, the base editing fusion protein as described in any one of items 5 to 9, or the base editing system as described in item 10 or 11 in the base editing of the chicken genome.

[0027] 23. The use of the cytosine deaminase as described in item 1, the base editing fusion protein as described in any one of items 5 to 9, or the base editing system as described in item 10 or 11 in the preparation of products for base editing of the chicken genome.

[0028] 24. The use of the cytosine deaminase as described in item 1, the base editing fusion protein as described in any one of items 5 to 9, or the base editing system as described in item 10 or 11 in the preparation of reagents for mediating base editing of the chicken genome.

[0029] Beneficial effects: This application extensively collected and integrated datasets from published literature, which contained sequence information and related biological characteristics of multiple known enzymes. Based on these datasets, cytosine deaminases from various sources were screened and their sequences and functions were compared. Combining structural prediction and functional verification, a cytosine deaminase derived from *Actinomyces biskii* was successfully screened and identified. Actinopolyspora biskrensis A cytosine deaminase (ch-YZ88-CBE) suitable for chicken cells and capable of efficient C-to-T editing was developed, and a CBE system with good editing activity and stability was constructed, providing key technical support for avian genetic improvement and functional gene research.

[0030] Compared with existing technologies, the ch-YZ88-CBE provided in this application effectively overcomes the technical bottlenecks such as the low editing efficiency of traditional cytosine deaminases in avian cells. It exhibits significantly enhanced activity in chicken cells, realizing efficient and precise C-to-T site-directed editing of the avian genome, and providing strong key technical support for avian genetic improvement and functional gene research. Attached Figure Description

[0031] The accompanying drawings are provided to better understand this application and do not constitute an undue limitation thereof. Wherein: Figure 1The predicted structure of ch-YZ88-CBE is shown. Figure 2 The diagram shows the sequencing peaks of sgRNA1 C-to-T mutation efficiency in DF-1 cells; Figure 3 The graph showing the mutation efficiency of sgRNA1 C-to-T in DF-1 cells is presented, and was evaluated using BEAR. Figure 4 The diagram shows the sequencing peaks of sgRNA2 C-to-T mutation efficiency in DF-1 cells; Figure 5 The graph showing the mutation efficiency of sgRNA2 C-to-T in DF-1 cells is presented, and was evaluated using BEAR. Figure 6 The graph showing the mutation efficiency of sgRNA3 C-to-T in DF-1 cells is presented, and was evaluated using BEAR. Figure 7 The diagram shows the sequencing peaks of sgRNA4 C-to-T mutation efficiency in DF-1 cells; Figure 8 The graph showing the mutation efficiency of sgRNA4 C-to-T in DF-1 cells is presented, evaluated using editR. Figure 9 The results show the quantitative results of the editing efficiency of sgRNA1 electrotransfection using the ch-SBG6-CBE base editor in DF-1 cells, which were evaluated using BEAR. Figure 10 The results show the quantitative results of the editing efficiency of sgRNA2 electrotransfection using the ch-SBG6-CBE base editor in DF-1 cells, which were evaluated using BEAR. Figure 11 The results show the quantitative results of the editing efficiency of sgRNA1 electrotransfection using the ch-PTEVA-CBE base editor in DF-1 cells, which were evaluated using BEAR. Figure 12 The results show the quantitative results of the editing efficiency of sgRNA2 electrotransfection using the ch-PTEVA-CBE base editor in DF-1 cells, which were evaluated using BEAR. Figure 13 The results show the quantitative results of the editing efficiency of sgRNA3 electrotransfection using the AXL9 base editor in DF-1 cells, which were evaluated using BEAR. Figure 14 The results show the quantitative results of the editing efficiency of sgRNA4 electrotransfection using AXL9 base editor in DF-1 cells, which were evaluated using editR; Figure 15 The results show the quantitative results of the editing efficiency of sgRNA3 electrotransfection using the AXT6 base editor in DF-1 cells, which were evaluated using BEAR; Figure 16 The results show the quantitative results of the editing efficiency of sgRNA4 electrotransfection using the AXT6 base editor in DF-1 cells, which were evaluated using editR; Figure 17 The results show the quantitative results of the editing efficiency of sgRNA3 electrotransfection using the BA88 base editor in DF-1 cells, which were evaluated using BEAR. Figure 18 The results show the quantitative results of the editing efficiency of sgRNA4 electrotransfection using the BA88 base editor in DF-1 cells, which were evaluated using editR; Figure 19 The results are quantifications of the editing efficiency of sgRNA3 electrotransfection using the ch-TU7-CBE base editor in DF-1 cells, evaluated using BEAR.

[0032] Figure 20 The results are quantifications of the editing efficiency of sgRNA4 electrotransfection using the ch-TU7-CBE base editor in DF-1 cells, evaluated using BEAR.

[0033] Figure 21 The results show the quantitative results of the editing efficiency of sgRNA1 by electrotransfection with BE4max in DF-1 cells, which were evaluated using BEAR; Figure 22 The results show the quantitative results of the editing efficiency of sgRNA2 by electrotransfection with BE4max in DF-1 cells, which were evaluated using BEAR. Detailed Implementation

[0034] The following description provides exemplary embodiments of this application, including various details to aid understanding, and should be considered merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0035] In this application, "base-editing fusion protein" or "fusion protein" refers to a protein that can mediate the substitution of one or more nucleotides at a target site in the genome in a sequence-specific manner. The one or more nucleotide substitutions are, for example, C-to-T substitutions.

[0036] In this application, "cytosine deaminase" refers to a deaminase that can accept nucleic acids, such as single-stranded DNA, as a substrate and can catalyze the deamination of cytidine or deoxycytidine into uracil or deoxyuracil, respectively.

[0037] In this application, a "nucleic acid targeting domain" refers to a domain capable of mediating the attachment of the base-editing fusion protein to a specific target site in the genome in a sequence-specific manner (e.g., via guide RNA). In some embodiments, the nucleic acid targeting domain may include one or more zinc finger protein domains (ZFP) or transcription factor effector domains (TALE) targeting a specific target site. In some embodiments, the nucleic acid targeting domain contains at least one (e.g., one) CRISPR effector protein.

[0038] In this application, the "zinc finger desmin domain (ZFP)" typically contains 3-6 individual zinc finger repeat sequences, each of which can identify a unique sequence of, for example, 3 bp. By combining different zinc finger repeat sequences, different genomic sequences can be targeted.

[0039] In this application, the "transcription activator-like effector domain" is the DNA-binding domain of a transcription activator-like effector (TALE). TALEs can be engineered to bind to virtually any desired DNA sequence.

[0040] In this application, "CRISPR effector protein" generally refers to a nuclease (CRISPR nuclease) or a functional variant thereof that exists in the naturally occurring CRISPR system. The term encompasses any CRISPR-based effector protein capable of sequence-specific targeting within cells.

[0041] In this application, the term "functional variant" in relation to a CRISPR nuclease means that it at least retains the guide RNA-mediated sequence-specific targeting ability. Preferably, the functional variant is a nuclease-inactivating variant, i.e., it lacks double-stranded nucleic acid cleavage activity. However, CRISPR nucleases lacking double-stranded nucleic acid cleavage activity also include nickases, which form a nick in a double-stranded nucleic acid molecule but do not completely cleave the double-stranded nucleic acid. In some preferred embodiments of this application, the CRISPR effector protein described herein has nickase activity. In some embodiments, the functional variant recognizes a different PAM (pre-intermediate sequence adjacent motif) sequence relative to the wild-type nuclease.

[0042] In this application, the "CRISPR effector protein" can be derived from a Cas9 nuclease, including a Cas9 nuclease or a functional variant thereof. The Cas9 nuclease can be a Cas9 nuclease from a different species, such as that from Streptococcus pyogenes (…). S.pyogenes spCas9 or derived from Staphylococcus aureus ( S.aureus SaCas9; "CRISPR effector proteins" can also be derived from Cpf1 nucleases, including Cpf1 nucleases or functional variants thereof. The Cpf1 nuclease can be a Cpf1 nuclease from a different species, for example, from... Francisella novicida U112 Acidaminococcus sp.BV3L6 and Lachnospiraceae bacterium The Cpf1 nuclease of ND2006. Available "CRISPR effector proteins" can also be derived from nucleases such as Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, Csn2, Cas4, C2c1 (Cas12b), C2c3, C2c2, Cas12c, Cas12d (i.e., CasY), Cas12e (i.e., CasX), Cas12f (i.e., Cas14), Cas12g, Cas12h, Cas12i, Cas12j (i.e., CasΦ), Cas12k, Cas12l, and Cas12m, including, for example, these nucleases or their functional variants.

[0043] In this application, "Cas9," "Cas9 nuclease," or "Cas9 domain" refers to CRISPR-associated protein 9 or variants thereof, including any natural Cas9 from any organism, any natural Cas9 equivalent or fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, and any variant of any naturally occurring or engineered Cas9. The term Cas9 is not limited to any specific Cas9 and may be referred to as "Cas9 or variants thereof." An exemplary Cas9 protein is described herein.

[0044] As used herein, the terms “Cas9” or “Cas9 nuclease” or “Cas9 domain” refer to CRISPR-associated protein 9 or variants thereof, including any native Cas9 from any organism, any native Cas9 equivalent or fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, and any variant of any naturally occurring or engineered Cas9. The term Cas9 is not limited to any specific Cas9 and may be referred to as “Cas9 or variants thereof.” Exemplary Cas9 proteins are described herein.

[0045] As used herein, the term "dCas9" refers to Cas9 with nuclease inactivation or death, or a variant thereof, and includes any naturally occurring dCas9 from any organism, any naturally occurring dCas9 equivalent or functional fragment thereof, any dCas9 homolog, ortholog, or paralog from any organism, and any naturally occurring or engineered dCas9 variant. Exemplary dCas9 proteins will be described herein. Any suitable mutation that inactivates the Cas9 nuclease can be used to form dCas9, such as the D10A and H840A mutations in the amino acid sequence of wild-type Streptococcus pyogenes Cas9, or the D10A and N580A mutations in the amino acid sequence of wild-type Staphylococcus aureus Cas9.

[0046] As used herein, the terms “nCas9,” “Cas9 cleavage enzyme,” or “Cas9 cleavage enzyme” refer to Cas9 or a variant thereof that cleaves only one strand, thereby introducing a gap in the double-stranded DNA molecule rather than creating a double-strand break. This can be achieved by introducing appropriate mutations into wild-type Cas9, such as the D10A or H840A mutation in the amino acid sequence of wild-type Streptococcus pyogenes Cas9, or the D10A mutation in the amino acid sequence of wild-type Staphylococcus aureus Cas9.

[0047] In this application, a "base editing system" refers to a combination of components required for base editing of nucleic acid sequences, such as genomic sequences in cells or organisms. The individual components of said system, such as cytosine deaminase, base editing fusion proteins, and one or more sgRNAs, may exist independently or in any combination as a composition.

[0048] In this application, "SgRNA," "guide RNA," and "CRISPR guide sequence" are used interchangeably, all referring to a type of nucleic acid containing a specific sequence that determines the targeting specificity of CRISPR effector proteins. SgRNA can hybridize with target sites in the host cell genome (hybridization can be partial or complete complementarity). The length of the SgRNA or its hybrid fragment hybridizing with the target site can range from 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides; in some embodiments, the length of the hybridization sequence can be specifically 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides; in other embodiments, its length ranges from 10-30 nucleotides or 15-25 nucleotides. It should be noted that SgRNA can be either an RNA sequence or a corresponding DNA sequence.

[0049] In this application, a "guide sequence" refers to a sequence within the guide RNA that is complementary to the target site and serves to guide the guide RNA to the target site for binding or modification (e.g., cleavage) by an RNA-guided DNA binder. The "guide sequence" may also be referred to as a "guide region," "targeting sequence," "spacer sequence," or "protospacers sequence." The guide sequence can be approximately 20 nucleotides in length.

[0050] In this application, gRNA, guide sequence, and gRNA scaffold can be an RNA sequence or a DNA sequence or DNA region corresponding to the RNA sequence.

[0051] In this application, "nucleic acid," "nucleic acid sequence," "nucleotide sequence," "polynucleotide," "polynucleotide sequence," "RNA sequence," or "DNA sequence" refers to oligonucleotides, nucleotides, or polynucleotides and their fragments or portions, and refers to DNA or RNA of genotype or synthetic origin, which may be single-stranded or double-stranded and represent sense or antisense strands. Sequences may be non-coding sequences, coding sequences, or mixtures of both. The nucleic acid sequences of this application can be prepared using standard techniques well known to those skilled in the art. Nucleotides are designated by their individual letter names as follows: "A" for adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), "C" for cytidine or deoxycytidine, "G" for guanosine or deoxyguanosine, "U" for uridine, "T" for deoxythymidine, "R" for purine (A or G), "Y" for pyrimidine (C or T), "K" for G or T, "H" for A, C, or T, "I" for inosine, and "N" for any nucleotide.

[0052] In this application, the percentage of "identity," such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%, refers to the degree of similarity between amino acid sequences or nucleotide sequences determined by sequence alignment, which is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%. For example, after introducing vacancies or other methods to make two sequences have as many identical residues as possible, the percentage of positions with identical bases or amino acid residues is determined relative to the total number of positions. The percentage of "identity" can be determined using software programs known in the art. It is preferred to use default parameters for alignment. A preferred alignment program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found at the following internet address: blast.ncbi.nlm.nih.gov / Blast.cgi.

[0053] In this application, the term "nuclear localization signal or sequence (NLS)" is an amino acid sequence that marks, designates, or otherwise tags a protein to be transported into the cell nucleus via nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysine or arginine residues exposed on the protein surface. Different nuclear localization proteins may share the same NLS. NLS have the opposite function to nuclear export signals (NES), which target proteins outside the cell nucleus. Thus, a single nuclear localization signal can guide the entity it is associated with into the cell nucleus. Such sequences can be of any size and composition, for example, exceeding 25, 25, 15, 12, 10, 8, 7, 6, 5, or 4 amino acids.

[0054] In this application, the term "uracil glycosylation enzyme inhibitor" or "UGI" refers to a protein that can inhibit uracil DNA glycosylation enzyme base excision repair enzyme.

[0055] In this application, "expression construct" refers to a vector, such as a recombinant vector, suitable for expressing a nucleotide sequence of interest in an organism. "Expression" refers to the production of a functional product. For example, the expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence (e.g., transcription to generate mRNA or functional RNA) and / or the translation of RNA into a precursor or mature protein. The "expression construct" of this application can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, a translatable RNA (e.g., mRNA). The "expression construct" of this application can contain regulatory sequences and nucleotide sequences of interest from different sources, or regulatory sequences and nucleotide sequences of interest from the same source but arranged in a manner different from those typically found naturally.

[0056] The terms "regulatory sequence" and "regulatory element" are used interchangeably. They refer to nucleotide sequences located upstream (5' non-coding sequence), midway, or downstream (3' non-coding sequence) of a coding sequence that influence the transcription, RNA processing, or stability or translation of the relevant coding sequence. Regulatory sequences may include, but are not limited to, promoters, translational leader sequences, introns, and polyadenylation recognition sequences.

[0057] In this application, "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell already introduced therein. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors".

[0058] In this application, "introduction" generally refers to the transfer of a foreign gene into recipient cells, such as eukaryotic or prokaryotic recipient cells. There are no particular limitations on the method of introduction; any known transformation method that can transfer the target gene into the recipient cell is acceptable. The method of introduction may include any of the following: (1) introducing the target gene or a recombinant vector containing the target gene into the host bacteria via chemical transformation (such as Ca ion-induced transformation, polyethylene glycol-mediated transformation, or metal cation-mediated transformation, etc.) or physical transformation (such as electroporation). (2) transducing the target gene into the host bacteria via bacteriophage transduction. (3) transferring the target gene into plant recipient cells via physical or chemical methods, such as gene gun method (also known as microparticle bombardment method or biological missile method), chemical stimulation method, electroporation method, liposome-mediated method, microinjection method, laser microbeam method, pollen tube channel method, ultrasound method, air gun method, and eddy current method, etc. (4) Using vectors to transfer the target gene into plant recipient cells, such as Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector system and binary vector system) mediated method (Agrobacterium-mediated method), plant virus vector mediated transformation method, etc.

[0059] In this application, the target site comprises a DNA sequence 5'-MCN-3', where M is A, T, C, or G, and N is A, T, C, or G.

[0060] As used herein, the term “in vitro” refers to experiments conducted in laboratory conditions or culture media using materials, biological substances, cells and / or tissues; while the term “in vivo” refers to experiments and procedures conducted using intact multicellular organisms.

[0061] As used herein, the term "about" or "approximately," when applied to one or more values ​​of interest, refers to a value similar to a specified reference value, or a value within an acceptable margin of error for a particular value as determined by a person skilled in the art, which will depend in part on how the value is measured or determined, such as limitations of the measurement system. In some respects, the term "about" refers to a range of values ​​falling within (greater or less than) 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of a specified reference value in any direction, unless otherwise stated or clearly apparent from the context (unless the number exceeds 100% of the possible value). Alternatively, according to practice in the art, "about" may mean within 3 or greater than 3 standard deviations. Or, for example, with respect to biological systems or processes, the term "about" may mean within an order of magnitude of the value, preferably within 5 times, more preferably within 2 times. This application provides a cytosine deaminase derived from Bisquercetin (B. biskes). Actinopolyspora biskrensisIt is named ch-YZ88-CBE.

[0062] In one specific embodiment of this application, the amino acid sequence of the cytosine deaminase is shown in SEQ ID NO:1; SEQ ID NO:1 has the following amino acid sequence: MSVGDVVERLRQAREQIPSSRLAEAADTIDQIRYTLAELGSEQDDAVEAYACAERIAELVEQARQVDATVRGKIETYLAQIGGSSGTSEPAAPTVISTGSDSTSSGTSEPTSEFVADDSSRYPIEAQWAR PLLEPRVVVGSGRPTIGQAKINGRHIGEMRSGRDGLSERVDERVRAVGIRTRRQLGNHVEMKLASLLIDSQARHGEIVINNAPCGTEPGAFAGCDQHLERFLPSGYSLTVYGTTAQGRKFAQTYHGKASS The cytosine deaminase of this application also comprises fragments, derivatives, and analogs of the ch-YZ88-CBE. As used herein, the terms "fragment," "derivative," and "analyte" refer to proteins that substantially retain the same biological function or activity as the ch-YZ88-CBE. The protein fragments, derivatives, or analogs of this application may be proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or may be proteins having substituent groups in one or more amino acid residues, or proteins formed by fusing additional amino acid sequences to this protein sequence (such as leader sequences, secretory sequences, sequences used to purify this protein, or proteoprotein sequences, or fusion proteins). As defined in this application, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0063] The cytosine deaminase of this application further comprises, compared with the ch-YZ88-CBE, a number of (typically 1-20, more preferably 1-10, even more preferably 1-8, 1-5, 1-3, or 1-2) amino acid deletions, insertions, and / or substitutions, and / or the addition or deletion of one or more (typically up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and / or N-terminus. For example, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of the protein.

[0064] In one specific embodiment of this application, the cytosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0065] Furthermore, the nucleotide sequence encoding the cytosine deaminase is shown in SEQ ID NO:20; SEQ ID NO:20 has the following nucleotide sequence: ATGAGCGTAGGAGATGTGGTGGAGAGGCTGAGGCAAGCACGGGAGCAGATCCCTTCCAGTCGATTAGCAGAGGCTGCGGACACCATTGATCAGATTCGTTACACCTTGGCAGAACTGGGTCAGAACAGGATGATGCAGTGGAGGCGTATGCCTGCGCTGAACGCATCGCTGAGCTGGTTGAACAAGCAAGGCAG GTTGATGCTACAGTGCGCGGGAAAATAGAGACGTATCTCGCCCAAATAGGTGGCTCCTCTGGCACCTCTGAGCCCGCTGCTCCCACAGTAATATCGACTGGGAGCGACTCTACTAGCAGTGGGACCTCCGAACCAACGTCAGAGTTTGTGGCCGACGACTCCAGCCGCTACCCAATTGAAGCACAGTGGGCCCGT CCGTTGCTTGAGCCTCGAGTCGTCGTTGGGAGCGGCAGACCAACAATCGGCCAGGCAAAAATTAATGGACGGCATATTGGGGAGATGAGAAGTGGTCGGGATGGACTCAGTGAGAGAGTGGATGAAAGGGTACGAGCCGTGGGTATCAGAACTAGACGTCAGCTGGGAAATCACGTGGAAATGAAGCTGGCCAGC TTACTTATTGACAGCCAAGCTAGACACGGAGAAATCGTCATCAACAACGCCCCTTGTGGAACAGAACCTGGTGCCTTTGCAGGTTGCGACCAGCACCTGGAGAGGTTTCCTCCCGTCAGGCTACTCTCTAACTGTTTATGGAACTACAGCTCAGGGCCGGAAGTTCGCGCAGACCTACCATGGGAAGGCATCATCC This application provides a base editing fusion protein containing a nucleic acid targeting domain and a cytosine deamination domain, wherein the cytosine deamination domain contains one or more of the cytosine deaminases provided in this application.

[0066] In one specific embodiment of this application, the nucleic acid targeting binding domain is selected from one or more of the following: CRISPR effector protein domains, zinc finger domains, and transcription activation-like effector factors.

[0067] In one specific embodiment of this application, the CRISPR effector protein is at least one of Cas9, Cpf1, Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, Csn2, Cas4, C2c1(Cas12b), C2c3, C2c2, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12l, Cas12m, or other available CRISPR effector proteins.

[0068] In one specific embodiment of this application, the CRISPR effector protein is Cas9, and the Cas9 is nuclease-inactivated Cas9, Cas9 nickase, or Cas9 with nuclease activity.

[0069] In one specific embodiment of this application, the Cas9 is a Cas9 nicking enzyme. Mutating and inactivating a subdomain of the DNA cleavage domain can endow Cas9 with nicking enzyme activity, i.e., obtain the Cas9 nicking enzyme.

[0070] In one specific embodiment of this application, the amino acid sequence of the Cas9 nickase is shown in SEQ ID NO:24; SEQ ID NO:24 has the following amino acid sequence: The nucleotide sequence encoding the Cas9 nickase is shown in SEQ ID NO:25.

[0071] In one specific embodiment of this application, the Cas9 nickase has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the amino acid sequence shown in SEQ ID NO:24.

[0072] In one specific embodiment of this application, the base-editing fusion protein comprises at least one nuclear localization sequence (NLS).

[0073] In one specific embodiment of this application, the NLS is located at the N-terminus and / or C-terminus of the base editing fusion protein. In some embodiments, the NLS is located between the cytosine deamination domain, the nucleic acid targeting domain, and / or the UGI.

[0074] In one specific embodiment of this application, the base editing fusion protein contains about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS at or near its C-terminus.

[0075] In one specific embodiment of this application, the base editing fusion protein contains about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS at or near its N-terminus; when there is more than one NLS, each NLS can be selected to be independent of the other NLS.

[0076] In one specific embodiment of this application, the base-editing fusion protein also has other localization sequences, such as cytoplasmic localization sequences or mitochondrial localization sequences.

[0077] In one specific embodiment of this application, the amino acid sequence of the base editing fusion protein is shown in SEQ ID NO:21; SEQ ID NO:21 has the following amino acid sequence: The nucleotide sequence encoding the base-editing fusion protein is shown in SEQ ID NO:22; In one specific embodiment of this application, the base-editing fusion protein has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the amino acid sequence shown in SEQ ID NO:21.

[0078] This application provides a base editing system for modifying chicken genes, comprising: a cytosine deaminase or a base editing fusion protein provided in this application and / or an expression construct containing a nucleotide sequence encoding the cytosine deaminase or the base editing fusion protein.

[0079] In one specific embodiment of this application, the base editing system further includes at least one sgRNA and / or at least one expression construct containing a polynucleotide sequence encoding the at least one sgRNA; it should be understood that if the base editing fusion protein is not based on a CRISPR effector protein, the base editing system does not require sgRNA or an expression construct encoding it.

[0080] In one specific embodiment of this application, the at least one sgRNA targets at least one target site in the chicken genome.

[0081] This application provides a method for base editing of the chicken genome, comprising: contacting the base editing system provided in this application with a target site in the chicken genome, thereby achieving base editing of the chicken genome.

[0082] In this application, the target site to be modified can be located anywhere in the chicken genome, such as within a functional gene like a protein-coding gene, or in a gene expression regulatory region such as a promoter region or an enhancer region, thereby achieving modification of the gene function or modification of gene expression.

[0083] In one specific embodiment of this application, the base editing system contacts the target site to perform deamination, resulting in the substitution of one or more nucleotides in the target site.

[0084] In one specific embodiment of this application, the deamination process leads to the introduction of a mutation in the gene promoter, which results in an increase or decrease in transcription of a gene operatively linked to the gene promoter.

[0085] In one specific embodiment of this application, the deamination process results in the introduction or removal of splice sites.

[0086] In one specific embodiment of this application, the deamination leads to the introduction of a mutation in the gene repressor, the mutation resulting in an increase or decrease in the transcription of a gene operatively linked to the gene repressor.

[0087] In one specific embodiment of this application, the contact takes place within the body.

[0088] In one specific embodiment of this application, the contact is performed outside the body.

[0089] This application provides a method for generating at least one modified chicken cell, comprising: introducing the base editing system provided in this application into at least one chicken cell, thereby causing substitution of one or more nucleotides at a target site in the at least one chicken cell.

[0090] In one specific embodiment of this application, the substitution of one or more nucleotides may be a C-to-T substitution. Furthermore, the base editing system can be introduced into chicken cells using any method known to those skilled in the art, such as calcium phosphate transfection, protoplast fusion, electrotransfection, liposome transfection, microinjection, viral infection, gene gun method, PEG-mediated protoplast transformation, or Agrobacterium-mediated transformation.

[0091] In one specific embodiment of this application, the method for generating at least one modified chicken cell provided in this application is performed in vitro, wherein the chicken cell is an isolated chicken cell or a chicken cell in an isolated tissue or organ.

[0092] This application provides a polynucleotide encoding the aforementioned uracil deaminase or base-editing fusion protein. Furthermore, the polynucleotide is codon-optimized, for example, for better expression in chicken cells.

[0093] This application provides a vector comprising the polynucleotide provided in this application.

[0094] In one specific embodiment of this application, the vector may be a cloning vector, an expression vector, a shuttle vector, an integration vector, a viral vector, a plasmid, a granule, or a bacteriophage.

[0095] This application provides a host cell, comprising the polynucleotide provided in this application and a vector. Further, the cell may be a prokaryotic cell or a eukaryotic cell.

[0096] In this application, the base editor is constructed by integrating the polynucleotide sequence of the base editing fusion protein provided in this application into an expression vector. Furthermore, other components of the editing editor are not limited and are all known in the art.

[0097] This application provides a reagent or kit for base editing of chicken genes, comprising the cytosine deaminase, the base editing fusion protein, or the base editing system provided in this application.

[0098] This application provides a composition comprising the cytosine deaminase, base-editing fusion protein, base-editing system, polynucleotide, vector, or base editor provided herein. Further, the composition further comprises a pharmaceutically acceptable vector.

[0099] This application provides the use of the cytosine deaminase described in the first aspect of this application, the base editing fusion protein described in the second aspect of this application, the base editing system described in the third aspect of this application, the polynucleotide described in the sixth aspect of this application, the vector described in the seventh aspect of this application, the base editor described in the ninth aspect of this application, or the composition described in the tenth aspect of this application in base editing of the chicken genome.

[0100] This application provides the use of the above-mentioned cytosine deaminase, base editing fusion protein, base editing system, polynucleotide, vector, base editor, or composition in the preparation of products for base editing of the chicken genome.

[0101] This application provides the use of the above-mentioned cytosine deaminase, base editing fusion protein, base editing system, polynucleotide, vector, base editor, or combination thereof in the preparation of reagents for mediating base editing of the chicken genome, including for reducing off-target effects, improving targeted editing efficiency, or improving the fidelity of targeted editing.

[0102] This application provides the use of the above-mentioned cytosine deaminase, base editing fusion protein, base editing system, polynucleotide, vector, base editor, or combination in the treatment of diseases in chickens.

[0103] The disease refers to a diagnosis of a point mutation-related or point mutation-caused disease, which can be corrected by the base editing fusion protein / base editing system of this application, for example, it can be used to correct any single-point T-to-C or A-to-G mutation.

[0104] This application provides the use of the above-mentioned cytosine deaminase, base editing fusion protein, base editing system, polynucleotide carrier, base editor, or composition in the preparation of a medicament for treating chicken diseases.

[0105] The disease referred to here means a disease diagnosed as being related to or caused by point mutations.

[0106] This application provides the use of the amino acid sequence shown in SEQ ID NO: 1 as a cytosine deaminase.

[0107] This application provides the use of the amino acid sequence shown in SEQ ID NO: 1 in the preparation of cytosine deaminase.

[0108] This application provides the amino acid sequence shown in SEQ ID NO: 1 as an application of cytosine deaminase in base editing of the chicken genome.

[0109] This application provides the amino acid sequence shown in SEQ ID NO: 1 as a cytosine deaminase for use in the preparation of products for base editing of the chicken genome.

[0110] Example The following description, in conjunction with specific embodiments, illustrates the content of this application, but the scope of this application is not limited thereto. Unless otherwise specified, the reagents and instruments used in the following embodiments are all conventional reagents and instruments in the art and can be obtained commercially. The methods used are all conventional experimental methods, and those skilled in the art can undoubtedly implement the described schemes and obtain corresponding results based on the embodiments.

[0111] Example 1 Screening of cytosine deaminases We extensively collected and integrated datasets from published literature, containing sequence information and related biological characteristics of multiple known enzymes. Based on these datasets, we conducted systematic evolutionary analysis to reveal the similarities and differences between different enzyme sequences and further screened enzymes exhibiting high catalytic activity as benchmarks. Building on this, we mined target sequences using protein databases, focusing particularly on novel sequences with a similarity of less than 70%. The core of this strategy is to ensure that the selected sequences are structurally highly differentiated, avoiding excessive similarity to existing known enzyme sequences, which could potentially lead to the omission of highly active enzymes. To ensure sequence quality and diversity, we placed particular emphasis on novelty and functional potential, selecting sequences with significant differences as the focus of subsequent analysis.

[0112] Subsequently, we applied the selected novel sequences to phylogenetic tree construction and further explored their similarities and differences in evolutionary relationships through cluster analysis. Through careful analysis of the clustering results, we removed redundant sequences to reduce the interference of repetitive information on the final screening results. To improve the quality of the final candidate deaminases, we further screened and predicted the functions of the remaining sequences after redundancy removal, ultimately identifying several potential high-activity deaminase sequences as candidates for subsequent experimental validation.

[0113] This embodiment, through further screening, obtained samples derived from *Bisk Actinopterygium* (…). Actinopolyspora biskrensis The single-base DNA deaminase (SddA) was named ch-YZ88-CBE, based on a bioinformatics analysis of existing single-base DNA deaminase (SddA) data.

[0114] Example 2 Construction of a base editor The amino acid sequence of ch-YZ88-CBE was confirmed by amino acid sequence comparison, and its structure was predicted as follows: Figure 1 As shown. The amino acid sequence of the ch-YZ88-CBE is shown in SEQ ID NO:1.

[0115] The sequence of SEQ ID NO:1 is as follows: MSVGDVVERLRQAREQIPSSRLAEAADTIDQIRYTLAELGSEQDDAVEAYACAERIAELVEQARQVDATVRGKIETYLAQIGGSSGTSEPAAPTVISTGSDSTSSGTSEPTSEFVADDSSRYPIEAQWAR PLLEPRVVVGSGRPTIGQAKINGRHIGEMRSGRDGLSERVDERVRAVGIRTRRQLGNHVEMKLASLLIDSQARHGEIVINNAPCGTEPGAFAGCDQHLERFLPSGYSLTVYGTTAQGRKFAQTYHGKASS ch-YZ88-CBE was synthesized and cloned into the AncBE4max vector with APOBEC removed by enzyme digestion to obtain the ch-YZ88-CBE base editor.

[0116] The nucleotide sequence of ch-YZ88-CBE is shown in SEQ ID NO:20; The fusion protein expressed by the ch-YZ88-CBE base editor has the amino acid sequence shown in SEQ ID NO:21; The fusion protein expressed by the ch-YZ88-CBE base editor has the amino acid sequence shown in SEQ ID NO:22.

[0117] Example 3 Construction of sgRNA expression vector Based on chicken ( Gallus gallus. Based on the NCBI classification number 9031 gene sequence, and according to the PAM sequence (NGG), the target region of the sgRNA for gene editing was selected, and two sgRNA sequences targeting the chicken gene were designed and synthesized, as follows: sgRNA1-F sequence: 5'-ccggTAACATGCAGACCAGACAGG-3' (SEQ ID NO: 2); sgRNA1-R sequence: 5'-aaacCCTGTCTGGTCTGCATGTTA-3' (SEQ ID NO: 3); sgRNA2-F sequence: 5'-ccggCATCCGGGTGCAGCCGGTGC-3' (SEQ ID NO: 4); sgRNA2-R sequence: 5'-aaacGCACCGGCTGCACCCGGATG-3' (SEQ ID NO: 5); sgRNA3-F sequence: 5'-ccggAAACCAAGGGACACAGATCA-3' (SEQ ID NO: 6); sgRNA3-R sequence: 5'-aaacTGATCTGTGTCCCTTGGTTT-3' (SEQ ID NO: 7); sgRNA4-F sequence: 5'-ccggACGGACAGACGGAGGCATGC-3' (SEQ ID NO: 8); sgRNA4-R sequence: 5'-aaacGCATGCCTCCGTCTGTCCGT-3' (SEQ ID NO: 9).

[0118] The DNA sequences of the four pairs of single-stranded sgRNAs were annealed to form two oligonucleotide chains of sgRNAs targeting different gene loci in chickens. Then, the four sgRNAs were ligated into the enzyme-digested linearized 74707 vector (Addgene, #74707) to obtain two expression vectors of sgRNAs targeting human nuclear genes.

[0119] Example 4: Editing Efficiency Test In this embodiment, after sequencing verification of the sgRNA expression vector constructed in Example 3, the plasmid was extracted and purified by sodium acetate precipitation. Using a ch-YZ88-CBE base editor to sgRNA ratio of 1:1, the ch-YZ88-CBE base editor and two purified plasmids were introduced into chicken embryo fibroblasts (DF-1) via electroporation. The culture medium was changed 12 hours after transfection, and 72 hours after transfection, the genome of each group of cells was extracted. PCR was performed using specific primers, and the obtained PCR products were sequenced using agarose gel electrophoresis. The editing status of specific gene sites in DF-1 cells was evaluated by analyzing the sequencing peak diagram.

[0120] The same PCR-specific primers are used for sgRNA1-F and sgRNA2-F, and the nucleotide sequences of the PCR-specific primers are shown in SEQ ID NO:10.

[0121] The sequence of SEQ ID NO:10 is as follows: GTTCATGTTAAGTGCCCGGAACG The same PCR-specific primers are used for sgRNA1-R and sgRNA2-R, and the nucleotide sequences of the PCR-specific primers are shown in SEQ ID NO:11.

[0122] The sequence of SEQ ID NO:11 is as follows: GTCATATCCTGGTTGTTAGCTGGC sgRNA3-F:GAGAGGAGTATGGTGATGACAGGG (SEQ ID NO: 12); sgRNA3-R:TAGCGGAGGGTAAGCACTCC (SEQ ID NO: 13); sgRNA4-F:ATGCAAGCCTTGTAGGAGCG (SEQ ID NO: 14); sgRNA4-R:TCTAAGCACACAAGCTAAGGAAGC (SEQ ID NO: 15).

[0123] The editing results of sgRNA1 are as follows Figures 2-3 As shown, the PAM sequence recognized by sgRNA1 is TGG. The mutation efficiency of C4-to-T was 27.8% and that of C8-to-T was 20.7%, as evaluated using BEAR software.

[0124] The editing results of sgRNA2 are as follows Figures 4-5As shown, the PAM sequence recognized by sgRNA2 is TGG, and the C5-to-T mutation efficiency was 16% as assessed by the website using BEAR software.

[0125] The editing results of sgRNA3 are as follows Figure 6 As shown, the PAM sequence recognized by sgRNA-3 is TGG. The mutation efficiency of C4-to-T was 13.7% and that of C5-to-T was 15.4%, as evaluated using BEAR software. The editing results of sgRNA4 are as follows Figures 7-8 As shown, the PAM sequence recognized by sgRNA-4 is TGG, and the mutation efficiency of C6-to-T is 16% as assessed by the website, using editR software. Comparative Example 1 This comparative example provides a cytosine deaminase obtained during the screening of cytosine deaminases as provided in Example 2, including ch-SBG6-CBE (database name A0A8U0SBG6 · A0A8U0SBG6_MUSPF), ch-PTEVA-CBE (database name A0A6P3RPW6 · A0A6P3RPW6_PTEVA), AXL9, AXT6, BA88, and ch-TU7-CBE. In this example, the inventors compared the activities of ch-YZ88-CBE, ch-SBG6-CBE, ch-PTEVA-CBE, AXL9, AXT6, BA88, and ch-TU7-CBE.

[0126] The amino acid sequence of AXL9 is shown in SEQ ID NO: 16. SEQ ID NO: 16 has the following amino acid sequence: MTGWVQLDGRDVGEITATRSDPWADDARRLLSLGLRRAKGLNNHVEMKTVTMMIQTNAREGRVIINHAPCGSEPGDPPGCDDVLPAFIPEGRTLTVLGTDAKGDPFKRTYHGKATR; The amino acid sequence of AXT6 is shown in SEQ ID NO: 17; SEQ ID NO: 17 has the following amino acid sequence: MPDRRTSDGVRDKTTGHVHHDDWPAIGDRRDTPVVSGYHPRWSAVAEQGLIEVGLRKRNARFLSAHVEIQVAAMMIATGRREVELTINHRPCPDNAPYLGCGSALPRYLPTGYTLTIYGTTIDGQPTVLRAQGQR; The amino acid sequence of BA88 is shown in SEQ ID NO: 18: MAIPDEVRRLRQVRNGLLVKRGWTRGVELKVAEIEAEAYRLLGDAARHSEVVRSLAEVRAKLDALREALGLAVDLVGQAIAHHSRTTGIGSSQASAAPRTIPPGPMSPVRGPAGAYVANQHGDLYPEEAIPY HEALQRRVVRGSGMPIAGWIQLDGAHIGEITATPNDPWAEASAERMADLDPRRAAFVAHHVEMKVIAMMVSLGSRHGQVIINHAPCGSEQGDPPGCHTSIPRCLPTGRTLTVLGTDRNGEPFTRTYHGKAKW The amino acid sequence of ch-TU7-CBE is shown in SEQ ID NO: 19: MGDDVAAVAARVREAMAKLPPEAFQIAGECIDEAGAGLHPLALETNDAELAAVIGALGDAREEIDRAWQICRKVRDACADYLKIIGAAEPSAPATSAGAAGGRARHVTAKDGSQCPPEAGAVVDVL PRRVREGEAGEKTVGFVDGSVTDKFVSGRDQTWTRSILARAREVGLPPHLARFVSSHVEMKVAAMMTQTGKQHCELVINHVPCGSQPAQPPGCDQAIERFLPKGYTLTVHGTTQASRPFSKTYRGQA Following the method in Example 2, ch-SBG6-CBE and ch-PTEVA-CBE base editors were constructed, respectively. Following the method in Example 4, the ch-SBG6-CBE and ch-PTEVA-CBE base editors, along with purified plasmids (sgRNA1 and sgRNA2), were electroporated into DF-1 cells. After transfection, PCR amplification was performed using specific primers. The amplification products were detected by agarose gel electrophoresis and then sequenced. Finally, the editing status of specific gene loci in DF-1 cells was evaluated using the sequencing peak diagram.

[0127] The editing result using the ch-SBG6-CBE base editor is as follows: Figures 9-10 As shown, its editing efficiency is significantly lower than that of the ch-YZ88-CBE base editor (such as...). Figure 3 and Figure 5 As shown in the figure, this indicates that ch-SBG6-CBE has weak activity.

[0128] The editing results using the ch-PTEVA-CBE base editor are as follows: Figure 11 , Figure 12 As shown, its editing efficiency is higher than that of the ch-SBG6-CBE base editor, but it is still significantly lower than that of the ch-YZ88-CBE base editor (e.g., Figure 3 and Figure 5 (As shown).

[0129] Following the method in Example 2, AXL9, AXT6, BA88, and ch-TU7-CBE base editors were constructed, respectively. Following the method in Example 4, the AXL9, AXT6, BA88, and ch-TU7-CBE base editors, along with purified plasmids (sgRNA3 and sgRNA4), were electroporated into DF-1 cells. After transfection, PCR amplification was performed using specific primers. The amplification products were detected by agarose gel electrophoresis and then sequenced. Finally, the editing status of specific gene loci in DF-1 cells was evaluated using the sequencing peak diagram.

[0130] The editing result using the AXL9 base editor is as follows: Figure 13 , Figure 14 As shown, its editing efficiency is significantly lower than that of the ch-YZ88-CBE base editor (such as...). Figure 3 and Figure 5 As shown in the image, AXL9 has almost no activity. The result of editing using the AXT6 base editor is as follows... Figure 15 , Figure 16 As shown, its editing efficiency is significantly lower than that of the ch-YZ88-CBE base editor (such as...). Figure 3 and Figure 5 As shown in the image, AXT6 has almost no activity. The result of editing using the BA88 base editor is as follows... Figure 17 , Figure 18 As shown, its editing efficiency is also significantly lower than that of the ch-YZ88-CBE base editor (such as...). Figure 3 and Figure 5 As shown in the image, BA88 has almost no activity. The editing result using the ch-TU7-CBE base editor is as follows... Figure 19 , Figure 20As shown, although its editing efficiency is improved compared to AXL9, AXT6, and BA88, it is still significantly lower than the ch-YZ88-CBE base editor (such as...). Figure 3 and Figure 5 (As shown).

[0131] The above results demonstrate that the ch-YZ88-CBE provided in this application successfully overcomes the limitations of existing cytosine deaminases in editing efficiency, providing strong key technical support for avian genetic improvement and functional gene research.

[0132] Comparative Example 2 In this comparative example, the efficiency of the ch-YZ88-CBE base editor and the BE4max base editor were compared. The amino acid sequence of the BE4max is shown in SEQ ID NO:23.

[0133] SEQ ID NO:23: MKRTADGSEFESPKKKRKVSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK Following the method in Example 2, a BE4max base editor was constructed. Using the method in Example 4, the BE4max base editor, along with purified plasmids (sgRNA1 and sgRNA2), was electrotransfected into DF-1 cells. After transfection, PCR amplification was performed using specific primers, and the amplification products were sequenced for analysis. The results are as follows: Figure 21 , 22 As shown, its editing efficiency is significantly lower than that of the ch-TU7-CBE base editor (such as...). Figure 3 and Figure 5 As shown in the figure, this indicates that ch-SBG6-CBE has weak activity.

[0134] Although the embodiments of this application have been described above, this application is not limited to the specific embodiments and application fields described above. The specific embodiments described above are merely illustrative and instructive, and not restrictive. Those skilled in the art can make many other forms based on the guidance of this specification and without departing from the scope of protection of the claims of this application, and these are all within the scope of protection of this application.

Claims

1. The use of a base-editing fusion protein in any of the following: (1) Application in base editing of the chicken genome; (2) Application in the preparation of products for base editing of the chicken genome; (3) Application in the preparation of reagents for mediating base editing of the chicken genome; in, The base-editing fusion protein comprises a CRISPR effector protein structure or a cytosine deaminase, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. The application according to claim 1, wherein, The base-editing fusion protein also contains at least one uracil DNA glycosylation inhibitor.

3. The application according to claim 1 or 2, wherein, The base-editing fusion protein also contains at least one nuclear localization sequence.

4. The application of a base editing system in any of the following: (1) Application in base editing of the chicken genome; (2) Application in the preparation of products for base editing of the chicken genome; (3) Application in the preparation of reagents for mediating base editing of the chicken genome; in, The base editing system comprises a base editing fusion protein and / or an expression construct containing a nucleotide sequence encoding the base editing fusion protein; The base editing fusion protein comprises a CRISPR effector protein domain and a cytosine deaminase, the amino acid sequence of which is shown in SEQ ID NO:

1.

5. The application according to claim 4, wherein the base editing system further comprises at least one sgRNA or at least one expression construct containing a nucleotide sequence encoding the at least one sgRNA; The at least one sgRNA targets at least one site in the chicken genome.

6. Application of cytosine deaminase in chicken genome base editing, among which, The amino acid sequence of the cytosine deaminase is shown in SEQ ID NO:

1.

7. The application of cytosine deaminase in the preparation of products for base editing in chicken genes, wherein, The amino acid sequence of the cytosine deaminase is shown in SEQ ID NO:

1.

8. The application of cytosine deaminase in the preparation of reagents for mediating base editing in the chicken genome, wherein, The amino acid sequence of the cytosine deaminase is shown in SEQ ID NO: 1.

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