Difunctional deaminase with adenine and cytosine bidirectional deamination activity and mutant, screening method and application thereof

Abstract

The invention provides a bifunctional deaminase with adenine and cytosine bidirectional deamination activity as well as a mutant, a screening method and application of the bifunctional deaminase. The amino acid sequence of the bifunctional deaminase comprises any one of the following sequences: (1) an amino acid sequence as shown in SEQ ID NO: 2; (2) an amino acid sequence which is derived from the amino acid sequence as shown in SEQ ID NO: 2 through substitution, deletion or addition of one or more amino acids and has adenine deamination activity and cytosine deamination activity; and (3) an amino acid sequence which has at least 80% identity with the amino acid sequence as shown in SEQ ID NO: 2 and has adenine deamination activity and cytosine deamination activity. The single-structural-domain bifunctional deaminase variant with high activity and balanced bidirectional deamination capacity is obtained through directed evolution, the editing activity of the variant is remarkably improved, bidirectional editing can be achieved only through a single polypeptide chain, and the bidirectional deamination capacity is balanced.

Description

[0001] This application claims priority to patent application number 2026101285400 (the earlier application was filed on January 29, 2026, entitled "A bifunctional deaminase with adenine and cytosine dual deamination activities, its mutants and applications"). Technical Field

[0002] This invention belongs to the field of biotechnology and gene editing, specifically relating to a bifunctional deaminase with dual deamination activities of adenine and cytosine, its mutants, screening methods, and applications. Background Technology

[0003] Precise genome editing is a core technology for understanding gene function, treating hereditary diseases, and improving crop genetics. Early gene editing tools, such as macronucleases, zinc finger nucleases (ZFNs), and transcription activation-like effector nucleases (TALENs), cut specific DNA sequences through protein-DNA specific recognition. However, due to their reliance on protein engineering for reorientation, they were complex to design, costly to construct, and difficult to apply at high throughput.

[0004] The discovery of the CRISPR-Cas system, especially the application of the Cas9 nuclease, has revolutionized the field of gene editing by enabling the targeting of specific sites in the genome through the simple RNA-DNA base pairing principle. Traditional CRISPR-Cas editing usually relies on introducing DNA double-strand breaks (DSBs) at the target site and then completing the rewriting through endogenous non-homologous end joining (NHEJ) or homologous recombination repair (HDR) pathways: (1) Although NHEJ is highly efficient, it often produces uncontrollable insertions / deletions (indels), which can easily lead to frameshifts and loss of function within open reading frames; (2) HDR depends on homologous templates, is less efficient, and is mainly active in dividing cells, making it difficult to fully play its role in many application scenarios; (3) Excessive DSBs may also induce genome rearrangements, large fragment deletions, and p53-mediated DNA damage responses, bringing potential safety risks. Therefore, developing precise base rewriting technology that does not rely on DSBs has become an important direction in the field of gene editing.

[0005] To overcome the problems caused by DSB, researchers developed a base editor (BE), which achieves the chemical conversion of specific bases without cutting double-stranded DNA by fusing inactivated or partially inactivated Cas proteins with base deaminases.

[0006] Cytosine base editor (CBE): CBE uses cytosine deaminases (such as rAPOBEC1 and A3A) as its core to deaminate cytosine (C) to uracil (U), which is then read as thymine (T) during replication or repair. Base transitions. Typical systems such as BE3 / BE4 achieve high efficiency through the APOBEC1-nCas9-UGI combination, but are prone to flanking C editing, C→A / C→G transversions, and a certain proportion of insertions / deletions (indels).

[0007] Adenine base editor (ABE): Adenine deaminase, which uses DNA as a substrate, is lacking in nature. Existing research has used... E. coli TadA (hereinafter referred to as ecTadA) underwent directed evolution to obtain a variant that can function on single-stranded DNA. And it integrates with nCas9 to build ABE7.10, achieving It features highly efficient base conversion; subsequently, a series of tools such as ABE8e and ABE9 were developed, further improving editing efficiency and compatibility.

[0008] Bifunctional base editors: Single CBEs or ABEs can only catalyze one type of base transition, which limits their application when simultaneously rewriting complex A and C mutation sites or constructing a fully covered saturated mutation library on the same target region. To address this, previous work has developed bifunctional base editors such as A&C-BEmax, Target-ACE / ACEmax, and SPACE by tandemly linking APOBEC family cytosine deaminases with ABE7.10 / ABE8e to the same nCas9 protein, achieving simultaneous C-to-T and A-to-G editing at the same target site.

[0009] However, these bifunctional editors with two deaminases in tandem have several common problems: (1) Large protein size and limited delivery: The fusion protein consisting of one Cas protein and two deaminase domains has a large molecular weight, which is not conducive to being loaded into adeno-associated virus and other capacity-limited vectors, and increases the host expression burden.

[0010] (2) Difficulty in balancing bifunctional editing activity: Different deaminases have different substrate ranges, editing windows, and optimal working conditions. Often, one type of editing is significantly stronger while the other is relatively weaker, making it difficult to obtain balanced A / C editing within the same window.

[0011] Against this backdrop, it is of great significance to develop novel deaminase chassis based on TadA homologs from different species, especially enzymatic modules that can simultaneously achieve A-to-G and C-to-T dual-function editing within a single domain.

[0012] With the widespread application of ABE (Alternating Base Editing), researchers have begun to focus on TadA orthologs from sources other than ecTadA. Existing work has screened and engineered various TadA homologs, introducing mutations homologous to ecTadA A106V / D108N into their respective backbones to construct ABEs, CBEs, and bifunctional base editors centered on TadA homologs. Some homologs have exhibited high initial editing activity after the introduction of specific mutations, indicating that the TadA family has the potential to be developed into multi-purpose base editing modules. Granulicella tundricola TadA (hereinafter referred to as gtTadA) is considered to have good A / C dual-function editing potential.

[0013] Overall, current research largely focuses on introducing a few homologous mutations into different TadA homologs to verify whether they possess ABE / CBE functions, lacking in-depth directed evolution and systematic engineering modification for specific TadA homologs. In particular, there is currently no publicly available mature technical solution for simultaneously obtaining efficient and relatively balanced DNA adenine and cytosine deamination activities on a single TadA homolog backbone through evolutionary means. Therefore, screening a single-domain bifunctional deaminase variant with high activity and balanced bidirectional deamination capabilities holds significant application potential.

[0014] MutaT7 is an in vivo directed mutagenesis tool based on a chimeric deaminase-T7 RNA polymerase (T7RNAP) protein. This system utilizes T7RNAP's specific recognition of the T7 promoter, targeting the carried deaminase to gene regions containing the T7 promoter. During transcription, the resulting transcription vesicle exposes non-template DNA as single-stranded DNA, which then becomes the substrate for the deaminase, enabling high-frequency base mutations in specific DNA fragments.

[0015] In the published literature, MutaT7 and its derivative systems are mainly used as an in vivo mutagenesis tool, that is, to regard deaminase-T7RNAP as a pre-defined mutation module, to continuously and directionally evolve downstream target genes (such as antibiotic resistance genes, key enzymes in metabolic pathways, etc.), and to obtain functionally optimized variants through growth selection or phenotypic screening.

[0016] Currently, there is no systematic evolution of the MutaT7 system for the deaminase-T7RNAP module itself, that is, using deaminase activity or editing profile as direct screening indicators to carry out large-scale directed evolution of deaminase domains. In particular, there is a lack of de novo optimization schemes for TadA homologs from non-E. coli sources. Summary of the Invention

[0017] To address the shortcomings of existing technologies, the present invention aims to provide a bifunctional deaminase with both adenine and cytosine deamination activities, its mutants, screening methods, and applications. This invention overcomes the limitations of existing physical fusion strategies for bifunctional deaminases and the deficiency of insufficient deamination efficiency of wild-type gtTadA on single-stranded DNA, providing a molecularly evolved single-domain deaminase variant.

[0018] To achieve this objective, the embodiments of the present invention employ the following technical solutions: In a first aspect, embodiments of the present invention provide a bifunctional deaminase having both adenine and cytosine deamination activities, wherein the amino acid sequence of the bifunctional deaminase includes any one of the following: (1) The amino acid sequence as shown in SEQ ID NO:2; (2) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO:2 by substitution, deletion or addition of one or more amino acids and having adenine deamination activity and cytosine deamination activity; (3) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO:2 (e.g., it may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, etc.) and has adenine deamination activity and cytosine deamination activity.

[0019] In this embodiment of the invention, the bifunctional deaminase shown in SEQ ID NO:2 is derived from... Granulicella tundricola The wild-type adenine deaminase was truncated, and the sequence after the 155th amino acid was removed.

[0020] Secondly, embodiments of the present invention provide a bifunctional deaminase mutant with dual deamination activities of adenine and cytosine, wherein the mutant is obtained by mutation based on the amino acid sequence shown in SEQ ID NO:2.

[0021] In some embodiments, the mutation site of the mutant is located at at least one of the following positions in the amino acid sequence shown in SEQ ID NO:2: position 102 or position 104.

[0022] In some embodiments, the mutation at position 102 of the mutant is selected from any one of A102V, A102I, or A102L.

[0023] In some embodiments, the mutation at position 104 of the mutant is selected from D104N or D104E.

[0024] In some embodiments, the mutation site of the mutant is selected from any one or a combination of at least two of A102V, A102I, A102L, D104N, or D104E.

[0025] In some embodiments, the mutation site is a combination of A102V and D104N; the mutated amino acid sequence is shown in SEQ ID NO:3.

[0026] In some embodiments, the mutant is obtained by directed evolution based on the mutant shown in SEQ ID NO:3, and the mutation sites in the directed evolution include any one or a combination of at least two of A13S, E19Q or I56V.

[0027] In some embodiments, the mutation sites in directed evolution are a combination of A13S, E19Q and I56V, and the mutated amino acid sequence is shown in SEQ ID NO:4.

[0028] In some embodiments, the mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO:4, and the mutation site of the mutant is located at position 45 of the amino acid sequence shown in SEQ ID NO:4.

[0029] In some embodiments, the mutation at position 45 of the mutant is selected from any one of I45V, I45L, I45A, or I45M.

[0030] In some embodiments, the mutation site is I45V, and the mutated amino acid sequence is shown in SEQ ID NO:5.

[0031] In this embodiment of the invention, conserved amino acid substitutions can be introduced at sites that substantially contribute to maintaining enzyme activity or stability. These conserved substitutions can be between amino acids with similar physicochemical properties, such as substitutions between nonpolar hydrophobic amino acids (e.g., I, V, L, M, A, etc.) or between acidic amino acids (D and E). Such substitutions typically do not significantly disrupt the protein's charge distribution and spatial folding stability, thereby contributing to maintaining the editing activity (e.g., C-to-T editing activity) and / or stability of the variant.

[0032] In this invention, I45V was confirmed as a key site for enhancing A-to-G activity. Based on the principle of protein hydrophobic core stacking and conserved substitution, replacing isoleucine (I) at position 45 with other nonpolar hydrophobic amino acids or amino acids with similar side chain volume (e.g., leucine L, alanine A, or methionine M) generally does not significantly disrupt the hydrophobic core stacking, thus it is expected to produce a structural fine-tuning effect similar to that of I45V. The variants obtained by the above substitution can have their bidirectional deamination activity and relative balance verified by the screening and quantitative evaluation system provided in this invention.

[0033] In this embodiment of the invention, the mutant SEQ ID NO:5 contains an amino acid substitution corresponding to the 45th position of the full-length gtTadA. This mutation is the decisive factor in conferring significant A-to-G editing activity to the variant and mediating the balance of bifunctional activities.

[0034] In this embodiment of the invention, the mutant shown in SEQ ID NO:4 was obtained by directed evolution based on the mutant shown in SEQ ID NO:3. Specifically, the mutant shown in SEQ ID NO:3 was used as the starting template for subsequent directed evolution to construct a mutant library and perform three rounds of directed evolution screening, including: specific screening for A-to-G editing, specific screening for C-to-T editing, and screening for both A-to-G and C-to-T editing respectively. After multiple rounds of iterative screening, the intermediate variant C25 (SEQ ID NO:4) was identified. Quantitative characterization showed that C25 had improved bidirectional activity compared to the starting template, but there was a significant activity bias: its C-to-T editing mutation rate was extremely high (reaching 10). -5 While the A-to-G editing mutation rate was higher than that of wild-type, it was still significantly lagging behind (only 10). -6 (in terms of scale), showing an unbalanced trend.

[0035] In this embodiment of the invention, the mutant shown in SEQ ID NO:5 was obtained through asymmetric evolution based on the mutant shown in SEQ ID NO:4. To address the activity imbalance problem of C25, an asymmetric evolution strategy was implemented. Using C25 as a template, a new round of ep-PCR libraries was constructed using the aforementioned random mutation kit. In the screening stage, only the library was transformed into the A-to-G editing reporter system, and two rounds of specific screening for A-to-G editing were performed consecutively. This prompted the enzyme to specifically search the evolutionary space for key mutations that could improve adenine deamination ability without restricting cytosine deamination activity, ultimately identifying the final variant C25-I45V (SEQ ID NO:5). After introducing the I45V mutation, the A-to-G editing activity of C25-I45V achieved a leap in efficiency (from 2.08 × 10⁻⁶). -6 Upgraded to 2.07 × 10-5 And it did not negatively affect C-to-T editing activity (maintained at 2.56 × 10⁻⁶). -5 The final variant achieved a mutation rate of 10 in both channels. -5 At a significant scale, a balance of dual-edit activity was successfully achieved.

[0036] In this embodiment of the invention, the mutant has the following advantages: (1) Significantly enhanced editing activity: In response to the problem that the wild-type or gtTadA variants with only a few rational mutations have low editing efficiency and cannot meet the needs of efficient genome editing, their catalytic efficiency and / or substrate affinity are improved through directed evolution, thereby significantly enhancing their editing activity to a level suitable for practical applications.

[0037] (2) Single domain dual function: without the need for tandem with other deaminase domains, efficient bidirectional editing of adenine (A-to-G) and cytosine (C-to-T) can be achieved through a single polypeptide chain, thereby significantly reducing the volume of the fusion protein and alleviating the problem of limited delivery by vectors such as adeno-associated virus.

[0038] (3) Balanced bidirectional deamination capacity: The device exhibits relatively balanced adenine and cytosine deamination activities on single-stranded DNA, overcoming the significant differences and biases in A-to-G and C-to-T editing activities present in existing bifunctional editors and some evolutionary intermediates. The balanced (or relatively balanced) bidirectional deamination capacity described in this embodiment can be characterized by the ratio of A-to-G to C-to-T editing activities (or mutation rates). In some embodiments, the bidirectional activity ratio is typically between 0.2 and 5, preferably between 0.5 and 2, and more preferably between 0.8 and 1.2.

[0039] (4) A novel enzymatic framework with differentiated features: an enzymatic framework not derived from ecTadA is used, breaking the existing ABE tool's high dependence on ecTadA and providing a deaminase chassis with different enzymatic properties and unique source.

[0040] Thirdly, embodiments of the present invention provide a nucleic acid base editing fusion protein, the fusion protein comprising: (a) The bifunctional deaminase with adenine and cytosine deamination activity as described in the first aspect or the bifunctional deaminase mutant with adenine and cytosine deamination activity as described in the second aspect.

[0041] In some embodiments, the fusion protein further includes at least one of the following elements: (b) Sequence-specific DNA-binding proteins; (c) Connector that connects (a) and (b).

[0042] In this embodiment of the invention, the fusion protein has the function of targeting the catalytic activity of deaminase to a specific DNA sequence, thereby achieving base editing at the target site.

[0043] In this embodiment of the invention, the sequence-specific DNA-binding protein is used to specifically recognize and bind to the target DNA sequence, serving as a localization module to guide the bifunctional deaminase to the intended genomic site.

[0044] In some embodiments, the linker is used to connect the deaminase domain to the DNA-binding protein and provides sufficient spatial degrees of freedom to prevent steric hindrance, ensuring that the two functional domains can function independently and synergistically.

[0045] In some embodiments, the XTEN Linker is used, and it can be replaced with a glycine-serine flexible linker (such as GS, GGGGS), a rigid linker (such as EAAAK), or a specially designed non-natural peptide to achieve different spatial folding requirements. As long as the linker can maintain the independent function of the deaminase and the localization module (such as T7RNAP), it can be used as an effective alternative component.

[0046] In some embodiments, the sequence-specific DNA-binding protein is selected from any one of: T7 RNA polymerase, CRISPR-Cas system effector proteins, or transcription activator-like effector proteins.

[0047] In some embodiments, the connector includes at least one of a flexible connector and a rigid connector.

[0048] In some embodiments, the CRISPR-Cas system effector protein includes any one of nCas9, dCas9, Cas12a, or Cpf1.

[0049] In some embodiments, the nucleic acid base editing fusion protein is a chimeric protein formed by fusing a bifunctional deaminase mutant with adenine and cytosine bidirectional deamination activity with T7 RNA polymerase, and is used to target the downstream region of the T7 promoter for continuous mutagenesis and saturation mutagenesis.

[0050] In some embodiments, the nucleic acid base editing fusion protein is in the form of N-terminal-TadA-Linker-T7 RNAP-C-terminus. This sequence can also be adjusted to N-terminal-T7 RNAP-Linker-TadA-C-terminus; furthermore, auxiliary elements such as nuclear localization signals (NLS), affinity tags (such as His-tag, Flag-tag), or uracil glycosylation inhibitors (UGI) can be inserted between the two without disrupting the catalytic active site of the deaminase.

[0051] In specific applications, non-catalytic components in fusion proteins can be omitted. Nuclear localization signals (NLS) and affinity tags only affect subcellular localization or purification methods and are not essential when the edited target does not require nuclear input or when performing in vitro reactions. While the omission of uracil glycosylase inhibitors (UGIs) may lead to reduced C-to-T editing efficiency or the production of byproducts due to interference from host base excision repair mechanisms, it does not alter the intrinsic biochemical activity of the deaminase variant itself in catalyzing cytosine deamination. Therefore, in directed evolution applications aiming to introduce diverse mutations using error-prone repair or in in vitro reaction systems without repair systems, the independent deaminase variant conformation without UGI fusion remains within the scope of this invention.

[0052] Fourthly, embodiments of the present invention provide a bifunctional base editor, the bifunctional base editor comprising: the nucleic acid base editing fusion protein described in the third aspect.

[0053] In some embodiments, the bifunctional base editor further includes guide RNA.

[0054] In this embodiment of the invention, the guide RNA is used to precisely guide gene editing tools (such as Cas9 protein or base editor) to a specific target location on the genome.

[0055] In some embodiments, the bifunctional base editor is formed by fusing a bifunctional deaminase mutant with adenine and cytosine bidirectional deamination activity with a sequence-specific DNA-binding protein (preferably a CRISPR-Cas system effector protein such as nCas9, dCas9, Cas12a, or TALE) for precise bifunctional editing of genomic sites.

[0056] Fifthly, embodiments of the present invention provide a nucleic acid molecule that encodes the bifunctional deaminase with adenine and cytosine deamination activities as described in the first aspect, a bifunctional deaminase mutant with adenine and cytosine deamination activities as described in the second aspect, or a nucleic acid base editing fusion protein as described in the third aspect.

[0057] Sixthly, embodiments of the present invention provide an expression vector comprising the nucleic acid molecule described in the fifth aspect.

[0058] In a seventh aspect, embodiments of the present invention provide a recombinant cell containing the expression vector described in the sixth aspect, or the genome of the recombinant cell is integrated with the nucleic acid molecule described in the fifth aspect.

[0059] Eighthly, embodiments of the present invention provide a kit containing the bifunctional deaminase with adenine and cytosine deamination activity as described in the first aspect, the bifunctional deaminase mutant with adenine and cytosine deamination activity as described in the second aspect, the nucleic acid base editing fusion protein as described in the third aspect, the bifunctional base editor as described in the fourth aspect, or the recombinant cells as described in the seventh aspect.

[0060] In a ninth aspect, embodiments of the present invention provide the application of the bifunctional deaminase having adenine and cytosine deamination activities as described in the first aspect, the bifunctional deaminase mutant having adenine and cytosine deamination activities as described in the second aspect, the nucleic acid base editing fusion protein as described in the third aspect, the bifunctional base editor as described in the fourth aspect, the recombinant cells as described in the seventh aspect, or the kit as described in the eighth aspect in the development of base editing or base editing tools.

[0061] In this embodiment of the invention, the application is, for example: Development of multiplex base editing tools: for preparing tools capable of co-mediating multiplex base editing at target sites in the genome of exogenous hosts (including prokaryotic or eukaryotic cell systems). and Gene editing systems involving base switching to achieve efficient construction of complex genotypes.

[0062] Construction of an in vivo continuous evolution system: Integrating into an in vivo evolution platform as a high-frequency mutagen. By introducing a highly diverse nucleotide variation profile into target nucleic acid sequences (such as key enzymes in metabolic pathways, antibody variable regions, or transcriptional regulatory elements), the system drives the functional optimization and adaptive evolution of target biomolecules.

[0063] In a tenth aspect, embodiments of the present invention provide a method for altering at least one nucleotide in a target DNA sequence, the method comprising: contacting the target DNA with the bifunctional base editor described in the fourth aspect to alter at least one nucleotide in the target DNA sequence.

[0064] Eleventhly, embodiments of the present invention provide a screening system comprising a nucleic acid molecule encoding the nucleic acid base editing fusion protein described in the third aspect.

[0065] In some embodiments, the screening system includes a bifunctional base editing element containing a nucleic acid molecule encoding the nucleic acid base editing fusion protein described in the third aspect.

[0066] In some embodiments, the screening system further includes a response element.

[0067] In some embodiments, the responsive element includes, but is not limited to, at least one of a plasmid vector, a phage vector, a Cos plasmid vector, an M13 phage vector, or a phage particle vector.

[0068] In some embodiments, the responsive element includes at least one of a C-to-T responsive element and an A-to-G responsive element.

[0069] In some embodiments, the screening system is used for quantitative screening and / or evolution of bifunctional deaminases.

[0070] In some embodiments, a screening system is provided, the system comprising: a bifunctional base editing plasmid and a reporter plasmid, the bifunctional base editing plasmid containing a nucleic acid molecule encoding the nucleic acid base editing fusion protein described in the third aspect; The reporter plasmids include: C-to-T active reporter plasmids and A-to-G active reporter plasmids; the reporter elements include: C-to-T response elements and A-to-G response elements.

[0071] In some embodiments, the C-to-T response element is a selectable marker gene containing a wild-type start codon mutated to an atypical start codon (such as ACG); the A-to-G response element is a selectable marker gene containing an early stop codon (such as TAG) introduced into the coding region.

[0072] In some embodiments, the C-to-T response element is a selectable marker gene containing a functional inactivation mutation, wherein the functional inactivation mutation is a mutation of the wild-type start codon to an atypical start codon (e.g., ACG), and the atypical start codon can be restored to a typical start codon by cytosine deamination.

[0073] In some embodiments, the A-to-G responsive element is a selectable marker gene containing a functional inactivation mutation, wherein the functional inactivation mutation is the introduction of an early stop codon (e.g., TAG) into the coding region, and the early stop codon can be repaired into a codon encoding an amino acid under the action of adenine deamination; the selectable marker gene is preferably the R67 dihydrofolate reductase gene.

[0074] In some embodiments, a reporter plasmid is provided to enable in-situ monitoring and substrate-specific differentiation of the catalytic activities of deaminase variants C-to-T and A-to-G.

[0075] Specifically, the reporter plasmid contains a genetically engineered R67 dihydrofolate reductase (DHFR) encoding nucleic acid sequence; this sequence introduces two functionally orthogonal genetic switches through site-directed mutagenesis to achieve independent decoupling and quantitative characterization of the bifunctional base deaminase activity: (1) Translation initiation recovery element: The wild-type start codon is mutated to an atypical ACG triplet (M1T, ATG→ACG). This site is configured as a conditional translation switch, which converts ACG to the canonical start codon ATG only under the action of cytosine deaminase (C-to-T) activity, thereby initiating the protein translation process.

[0076] (2) Premature termination elimination element: A nonsense mutation (e.g., W23Stop, TGG→TAG) is introduced inside the coding region. This site is configured as a read-through switch, which will only replace the stop codon with a codon encoding tryptophan (Trp) under the action of adenine deaminase (A-to-G) activity, thereby eliminating the premature termination signal of translation.

[0077] The above-mentioned construct utilizes a single cistron framework and changes in the trimethoprim (TMP) resistance phenotype to achieve in-situ monitoring and substrate-specific differentiation of the catalytic activities of two deaminase variants, C-to-T and A-to-G.

[0078] In this embodiment of the invention, to adapt to different host backgrounds or experimental needs, key components of the screening platform can be functionally equivalently replaced. R67 (DHFR) can be replaced with other screenable markers based on the principle of functional recovery, such as kanamycin (DHFR). Kan R ), chloramphenicol ( Cm R ) or tetracycline ( Tet R Resistance genes are identified by constructing screening plasmids through the introduction of early stop codons (TAG) or start codon mutations (ACG) into the active site. The corresponding screening pressure reagent, trimethoprim (TMP), can be replaced with antibiotics corresponding to the aforementioned reporter genes (such as G418, chloramphenicol, or bleomycin), or screening can be performed by correcting lethal mutations using toxin-antitoxin systems such as CcdB.

[0079] Alternatively, mutants of fluorescent protein genes such as GFP and RFP can be used to screen for positive cells with restored fluorescence signals via flow cytometry (FACS). Such substitutions do not affect the guidance and enrichment of enzymes in their evolutionary direction.

[0080] In a twelfth aspect, embodiments of the present invention provide a method for directed evolution or quantitative assessment of the activity of bifunctional deaminases using the screening system described in the eleventh aspect, the method comprising: co-transforming the expression element of the deaminase to be tested and the response element into host cells; and determining the editing activity of A-to-G or C-to-T by resistance screening and / or phenotypic analysis.

[0081] In some embodiments, the directed evolution includes an asymmetric screening step: (1) Provide a mutant library and transform it into a host cell containing the screening system described in the eleventh aspect; (2) In the first screening stage, the first editing activity was enriched by adding screening reagents to the culture medium; (3) In the second screening stage, the imbalance of bidirectional activity is corrected by switching the response element for the second editing activity or changing the screening window, and by specifically enriching the second editing activity in the presence of the screening reagent.

[0082] In some embodiments, in step (1), the response element includes at least one of a C-to-T response element and an A-to-G response element.

[0083] In some embodiments, the screening reagents in step (2) include, but are not limited to, trimethoprim.

[0084] In some embodiments, the asymmetric screening step further includes alternating between steps (2) and (3) until a bidirectional balanced variant is obtained.

[0085] The embodiments of this invention demonstrate that the C-terminal sequence of gtTadA is not essential for its deamination catalytic activity. Therefore, the scope of protection of this invention is not limited to the specific truncated length shown in SEQ ID NO:2, but covers all truncated variants that retain the N-terminal catalytic core (approximately amino acids 1-155) and possess deamination activity. Similarly, any fine-tuning of the ends of such truncated variants (e.g., extending or shortening by several amino acids) without disrupting the complete secondary structure folding of the N-terminal catalytic core falls within the scope of protection of this invention.

[0086] Compared to existing technologies, embodiments of the present invention construct based on Granulicella tundricola The novel TadA deaminase and its accompanying evolutionary platform have the following significant beneficial effects: (1) Achieve dual-function editing of a single structural domain and break through the bottleneck of virus vector delivery.

[0087] Existing dual-function editors often employ a strategy of physically tandemly linking adenine deaminase and cytosine deaminase, resulting in fusion proteins that are enormous (typically >400 aa), making them difficult to load into capacity-limited delivery vectors such as adeno-associated viruses. The C25-I45V variant provided in this invention is based on a truncated backbone of gtTadA-155N, requiring only a single domain of approximately 155 amino acids to simultaneously achieve efficient A-to-G and C-to-T editing. Compared to traditional dual-enzyme fusion systems, this design significantly reduces protein volume, effectively lowers the packaging burden of gene therapy vectors, and avoids the steric hindrance and stability issues that may be introduced by multi-domain fusion.

[0088] (2) Achieve a balanced A / C bidirectional editing capability.

[0089] To address the imbalance between A-to-G and C-to-T editing activities commonly found in existing tools, this invention employs a unique asymmetric evolutionary strategy to successfully identify the key variant C25-I45V. This variant, C25-I45V, maintains the high C-to-T editing activity of C25 while specifically enhancing A-to-G editing activity, thus possessing highly active and relatively balanced bidirectional deamination capabilities on single-stranded DNA, making it more suitable for handling complex compound mutation correction.

[0090] (3) Provide a differentiated enzymological framework to enrich the gene editing toolbox.

[0091] Current mainstream adenine base editors (ABE) are highly dependent on Escherichia coli The TadA backbone results in limited enzymatic properties. The gtTadA variant (core functional domain) developed in this invention shares only about 54% amino acid sequence similarity with ecTadA, classifying them as distant homologs. This provides a novel, differentiated, and autonomously controllable core enzyme element for the gene editing field, increasing the selectivity of base editing tools in terms of substrate preference and temperature adaptability.

[0092] (4) Establish a new evolutionary platform based on MutaT7.

[0093] This study expands the application boundaries of the MutaT7 system, transforming it from a conventional mutagenesis tool into a screening platform specifically designed for the evolution of deaminase modules themselves. By constructing R67 reporter systems sensitive to A-to-G and C-to-T editing respectively, independent characterization and quantitative assessment of two deaminase activities were achieved within the same bacterial host context. This platform not only successfully screened the gtTadA variant but also provides a standardized and universal screening strategy for the discovery and directed evolution of deaminases from other non-model organism sources. Attached Figure Description

[0094] Figure 1 This is a schematic diagram illustrating the evolutionary screening and quantitative characterization principle of the MutaT7-R67 bifunctional base editing system.

[0095] Figure 2 A flowchart for targeted evolution and alternating screening optimized for bifunctional activity.

[0096] Figure 3 This is a multiple sequence alignment diagram of key amino acids in deaminase variants.

[0097] Figure 4 Characterize the bifunctional editing activity and activity ratio of variants at different evolutionary stages.

[0098] Figure 5 A schematic diagram of the structure of a fusion protein for application in genome editing. Detailed Implementation

[0099] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0100] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0101] The experimental materials and general methods involved in the following specific implementation methods are shown below.

[0102] 1. Experimental materials.

[0103] Strains: Cloning construction using chemically competent cells E. coli DH5α (purchased from AlpalifeBio); chemicompetent cells were used for MutaT7 screening and activity characterization. E. coli DH10B (purchased from Biomed).

[0104] Enzymes and kits: PCR amplification was performed using 2×Phanta UniFi Master Mix (purchased from Vazyme, P516); colony PCR was performed using Premix Taq. TM DNA Polymerase (Takara, R901A); homologous recombination cloning was performed using the ClonExpress Ultra One Step Cloning Kit V3 (Vazyme, C117); error-prone PCR was used to construct mutant libraries using QuickMutation. TM Random Mutagenesis Kit (purchased from Beyotime, D0219S) and Random Mutagenesis Kit (purchased from Biosharp, BL1668A).

[0105] Plasmid backbone: The expression vector was modified from the pDae079 vector system (see Addgene Plasmid #187622 for details). This vector backbone carries the ampicillin resistance gene. Amp RThe original pDae079 contains two deaminase domains. After modification, it retains only a single deaminase component site for insertion into the TadA variant to construct the [TadA]-[XTENLinker]-[T7 RNAP] form of the MutaT7 fusion protein. The reporter plasmid is modified from a low-copy plasmid driven by the T7 promoter (containing a tandem T7 terminator array, see Plasmid #156456 for details). This vector backbone carries the chloramphenicol resistance gene. Cm R By replacing the original Tn5 source with R67. Neo R / Kan R It is constructed from genes and used for subsequent resistance recovery screening.

[0106] Primers and genes: All primers were synthesized by Genewiz; the R67 and TadA variant gene fragments were synthesized by General Bio.

[0107] Chemical reagents: ampicillin (Amp, 100 µg / mL), chloramphenicol (Cm, 25 µg / mL), trimethoprim (TMP, 20 µg / mL), L-arabinose (L-arabinose, 0.2% w / v); all the above reagents were purchased from commercially available sources.

[0108] 2. General methods.

[0109] General procedure for quantitative calculation of mutation rate.

[0110] To enable cross-study comparisons with other MutaT7-related studies and to quantitatively characterize the intracellular editing activity of deaminase variants using genetic data, this invention employs the Luria-Delbrück rare mutation approximation model, converting the endpoint phenotypic mutation frequency into per-base per-generation mutation rates. The specific steps are as follows: (1) Induced expression and mutation accumulation: The expression plasmid and reporter plasmid are co-transformed into competent cells (e.g., E . coliAfter DH10B, the culture was spread onto LB agar plates containing the corresponding antibiotics (100 µg / mL Amp and 25 µg / mL Cm) and incubated overnight (12-16 hours) at 37°C. The next day, single colonies were selected from the plates and inoculated into 10 mL of liquid LB medium supplemented with the corresponding antibiotics (100 µg / mL Amp and 25 µg / mL Cm) and 0.2% L-arabinose. The culture was incubated overnight (16 hours) at 37°C with shaking at 220 rpm to initiate TadA expression and mutation accumulation. The next day, the overnight culture was transferred at a ratio of 1:100 to fresh LB medium (containing the same concentrations of antibiotics and L-arabinose). To promote mutation fixation during active growth, the transferred culture was incubated at 37°C with shaking at 220 rpm for 4 hours.

[0111] (2) Data Acquisition: At the end of the culture, the bacterial culture was serially diluted (10-fold). 10 µL of each serial dilution was spotted onto non-selective plates (containing 100 µg / mL Amp and 25 µg / mL Cm), and single colonies were counted after incubation. Simultaneously, 300 µL of undiluted bacterial culture was spread onto selective plates (containing the above antibiotics and 20 µg / mL TMP), and single colonies were counted after incubation (three technical replicates were established). Based on the dilution factor and the spread / spotting volume, the original colony count on the plate was extrapolated to the total culture volume to obtain the total viable count of the induction system. N 0 ) and the total number of resistance-reverting mutant bacteria in the induction system ( N 1 The endpoint mutation frequency is calculated as follows: .

[0112] (3) Mutation rate conversion: In order to achieve cross-study comparison, the endpoint mutation frequency is converted into the mutation rate per base per generation. According to the Luria-Delbrück rare mutation approximation model, the expected frequency satisfies the formula. ,in μ That is, the mutation rate per base per generation mentioned above. This represents the natural logarithmic generation number. In the continuous culture protocol of this embodiment, although induction was maintained for 16 hours, the effective population expansion normalized to the final regrowth step was calculated because mutation fixation depends on replication. This single 4-hour passage includes 1:100 transfer and subsequent regrowth to saturation, corresponding to the number of generations. G Approximately 6.6 generations. Based on the bacterial binary fission hypothesis ( The effective normalization factor is calculated as follows: Since TMP resistance restoration in the R67 reporter system strictly depends on single-base reverse mutations, the measured frequency is directly considered as the site-specific mutation frequency, and the full-length 192 bp reporter gene is not normalized. Therefore, the formula for calculating the site-specific mutation rate is: .

[0113] The sequences involved in the following specific implementation are shown below.

[0114] SEQ ID NO:1 MTPDEQFLREAIAEARAAEQAGEVPVGAVLVLNNEIIARGRNRVILDSDPTAHAEIVALREAGRILGNYRLENCDLYTTLEPCAMCAGAILHARIRRLIYAAADPKAGACGSALDVMNHPRLNHRMEVAVGLLAEECGEMLTSFFR TRRLKNKENAASAIGIEALMTTVKKSAAPKKSAAKTTAKKTAAKPPHKWSAKVTTTDSTHPDEGLFNEDAQTIAKKLASKKVSPKGPASGMQMLNFYINRAGKNLPKARQAELEKAKDILSQIIADAKPKAPAKKAGRKTPAKKTAN.

[0115] SEQ ID NO:2 MTPDEQFLREAIAEARAAEQAGEVPVGAVLVLNNEIIARGRNRVILDSDPTAHAEIVALREAGRILGNYRLENCDLYTTLEPCAMCAGAILHARIRRLIYAAADPKAGACGSALDVMNHPRLNHRMEVAVGLLAEECGEMLTSFFRTRRLKNKEN.

[0116] SEQ ID NO:3 MTPDEQFLREAIAEARAAEQAGEVPVGAVLVLNNEIIARGRNRVILDSDPTAHAEIVALREAGRILGNYRLENCDLYTTLEPCAMCAGAILHARIRRLIYAVANPKAGACGSALDVMNHPRLNHRMEVAVGLLAEECGEMLTSFFRTRRLKNKEN.

[0117] SEQ ID NO:4 MTPDEQFLREAISEARAAQQAGEVPVGAVLVLNNEIIARGRNRVILDSDPTAHAEVVALREAGRILGNYRLENCDLYTTLEPCAMCAGAILHARIRRLIYAVANPKAGACGSALDVMNHPRLNHRMEVAVGLLAEECGEMLTSFFRTRRLKNKEN.

[0118] SEQ ID NO:5 MTPDEQFLREAISEARAAQQAGEVPVGAVLVLNNEIIARGRNRVVLDSDPTAHAEVVALREAGRILGNYRLENCDLYTTLEPCAMCAGAILHARIRRLIYAVANPKAGACGSALDVMNHPRLNHRMEVAVGLLAEECGEMLTSFFRTRRLKNKEN.

[0119] Example 1 The construction and rational transformation of the functional framework.

[0120] Granulicella tundricola The full-length sequence of TadA (gtTadA) is 292 aa (SEQ ID NO:1). Sequence alignment with ecTadA and superposition analysis using PyMOL software to compare the AlphaFold3-predicted gtTadA structure with the ecTadA crystal structure (PDB ID:6VPC) revealed that the N-terminus of gtTadA (approximately amino acids 1-155) contains a complete deaminase catalytic core fold, while the C-terminal sequence is not functionally essential. To construct an enzyme molecule more suitable as a gene editing tool, a truncated version, gtTadA-155N (SEQ ID NO:2), was designed, which is the sequence after amino acid 155 removed. Using homologous recombination, the full-length sequence and the truncated fragment were cloned into a modified expression vector backbone (based on pDae079, Addgene #187622, ​​modified to retain only a single deaminase component site), named pMutaT7-gtWT and pMutaT7-gt155, respectively.

[0121] Two key rational mutations, A102V and D104N, were introduced into the gtTadA-155N backbone using site-directed mutagenesis. This variant was named gtTadA-155N-VN (SEQ ID NO:3), and its corresponding expression plasmid was named pMutaT7-gt155VN, serving as the starting template for subsequent directed evolution.

[0122] In addition, a plasmid pMutaT7-ΔTadA was constructed that expresses only [XTEN Linker]-[T7RNAP] but lacks the deaminase module, as a negative control for the activity test.

[0123] Example 2 Establishment and validation of a dual-function quantitative screening platform.

[0124] A screening platform was constructed using a reporter plasmid (based on a T7 promoter + terminators reporter, Addgene #156456). The NeoR / KanR gene in the precursor plasmid (Addgene #156456) was replaced with the R67 gene fragment to construct the pReporter-R67 basic backbone, driven by the T7 promoter and containing a tandem T7 terminator array. Based on this, two independent screening platforms were constructed using site-directed mutagenesis: C-to-T active reporter plasmid (pReporter-R67) M1T The start codon ATG of the R67 gene is mutated to ACG. Functional R67 protein expression is only possible when the TadA variant restores ACG to ATG through C-to-T editing activity, thereby conferring TMP resistance to the host bacteria.

[0125] A-to-G activity reporter plasmid ( The TadA variant mutates the TGG codon encoding tryptophan at position 23 of the R67 gene to the stop codon TAG. Functional R67 protein expression, conferring TMP resistance, is only achieved when the TadA variant mediates A-to-G editing to repair TAG back to TGG.

[0126] To verify the effectiveness of the platform, the three expression plasmids pMutaT7-gtWT (full-length), pMutaT7-gt155 (truncated), and pMutaT7-gt155VN (evolutionary start template) were co-transformed with the aforementioned two reporter plasmids to [the desired platform]. E . coli DH10B.

[0127] Following the general procedure described in the "General Calculation of Mutation Rate" section of the universal method, the results showed that the mutation rate of pMutaT7-gt155VN was significantly higher than that of pMutaT7-gt155 and pMutaT7-gtWT on both screening platforms. Furthermore, the activities of all three were significantly higher than the negative control pMutaT7-ΔTadA (which showed no colony growth on TMP selective plates). These results demonstrate the successful construction of the bifunctional screening platform and its ability to support subsequent directed evolution screening. It also demonstrates that the gtTadA-155N-VN variant possesses higher bidirectional deamination activity and is more suitable as a template for directed evolution.

[0128] Figure 1 This is a schematic diagram illustrating the evolutionary screening and quantitative characterization principle of the MutaT7-R67 bifunctional base editing system. (A) shows the expression plasmid construction diagram, illustrating the process... P BAD (B) Promoter-induced expression of deaminase-T7 RNA polymerase fusion protein; (C) Reporter plasmid design, containing... P T7 (C) Promoter-driven R67 gene carrying key inactivation mutations (used to detect C-to-T and A-to-G editing, respectively); (C) Schematic diagram of resistance reversion screening and mutation frequency quantification based on base editing.

[0129] Example 3 Establishment of a general method for directed evolution.

[0130] This embodiment details the general library construction and screening process for the evolution of TadA variants.

[0131] 1. Preparation of competent cells containing reporter plasmids.

[0132] To ensure the transformation efficiency and library size of the mutant library, each library construction will include an A-to-G reporter plasmid on the same day. ) or C-to-T reporter plasmid (pReporter-R67) M1T )of E. coli DH10B cells were prepared into fresh electrocompetent cells according to standard procedures and immediately used for subsequent transformation of mutant libraries (without cryopreservation).

[0133] 2. Construction and import of mutant libraries.

[0134] Based on the starting template or plasmids enriched after the previous round of stress screening, error-prone PCR (ep-PCR) was performed using a random mutagenesis kit. The kit was operated strictly according to the recommended conditions, controlling the mutation rate to 1-3 amino acids / sequence. The PCR products were purified by Urea-PAGE denaturing polyacrylamide gel electrophoresis to remove primer dimers and non-specific amplification bands. After one round of conventional PCR amplification and enrichment, the purified products were homologously recombinated into the modified expression plasmid backbone. The purified recombinant products were then electroporated into freshly prepared competent cells containing reporter plasmids and subjected to liquid resuscitation culture. The calculated library size should be >10. 6 CFU is used to ensure diversity.

[0135] 3. Screening process and NGS sample preparation.

[0136] To accurately track evolutionary trajectories and eliminate background noise, embodiments of this invention employ a rigorous cleaning and screening process: (1) Library recovery and washing: The bacterial culture after electroporation was cultured overnight for 12 hours in LB medium containing Amp+Cm. The next day, the bacterial cells were collected by centrifugation and washed and resuspended in fresh antibiotic-free LB medium to completely remove residual antibiotics and dead bacterial precipitate.

[0137] (2) Pre-selection library preparation: The washed resuspension was inoculated into LB containing Amp+Cm and cultured overnight for 12 hours to allow the library to amplify to saturation. Plasmids were extracted from a portion of the bacterial cells. Using this plasmid as a template, the target gene fragment (TadA variant region) was amplified using specific NGS primers and a sequencing library was constructed, denoted as the pre-selection library, which was used for NGS analysis of initial diversity.

[0138] (3) Preparation of post-selection library: The above bacterial culture was transferred to selective medium containing TMP+Amp+Cm at a ratio of 1:100 and cultured at 37°C with shaking for 12 hours. The surviving resistant bacterial cells were collected and plasmids were extracted. The plasmids were amplified and constructed using NGS primers to serve as the post-selection library for evaluating enrichment.

[0139] Example 4 Multiple rounds of iterative screening and the acquisition of intermediate C25.

[0140] 1. Multiple rounds of iterative screening.

[0141] Using the general method established in Example 3, the first three rounds of directed evolution screening were performed while maintaining a TMP concentration of 20 µg / mL. The specific procedure is as follows: Round 1: First, convert the document library to the A-to-G editing platform ( The process involves screening the clones, eliminating inactive variants through the aforementioned pressure screening, and enriching clones with basic A deamination capabilities.

[0142] Second round: Extract plasmids from the libraries after the first round of screening and transform them into the C-to-T editing platform (pReporter-R67). M1T The process involves screening to further enrich highly active variants.

[0143] The third round: plasmids from the libraries after the second round of screening were extracted, transformed into two platforms in parallel for screening, and NGS libraries were prepared separately, resulting in a total of four sequencing libraries.

[0144] 2. Determination of intermediate C25.

[0145] High-throughput sequencing was performed on the libraries before and after the third round of screening, and the enrichment scores (Fitness Score) of each variant were analyzed using deep mutation scanning. Based on the data analysis results, several candidate variants that showed high enrichment during screening were selected for activity testing, and the intermediate variant C25 (SEQ ID NO:4, whose corresponding expression plasmid was named pMutaT7-C25) was identified. Quantitative characterization showed that C25 had enhanced activity in both directions compared to the starting template, but there was a significant activity bias: its C-to-T editing mutation rate was extremely high (reaching 10). -5 While the A-to-G editing mutation rate was higher than that of wild-type, it was still significantly lagging behind (only 10). -6 (in terms of scale), showing an unbalanced trend.

[0146] Example 5 Asymmetric evolution and the acquisition of the final variant C25-I45V.

[0147] To address the activity imbalance issue associated with C25, an asymmetric evolution strategy was implemented. Using C25 as a template, a new ep-PCR library was constructed using the aforementioned random mutation kit. During the screening phase, only the library was transformed into the A-to-G editing reporter system (A-to-G). )middle.

[0148] Figure 2 This is a flowchart illustrating the directed evolution and alternating screening process for bifunctional activity optimization. The diagram shows the complete experimental pathway from the initial template, through multiple rounds of error-prone PCR mutagenesis and alternating C-to-T / A-to-G screening, to first obtain the intermediate C25. Subsequently, addressing the activity bias of C25 (i.e., C-to-T editing activity is significantly higher than A-to-G editing activity), targeted A-to-G editing pressure screening (i.e., an asymmetric strategy) is employed to finally prepare the balanced variant C25-I45V.

[0149] 1. Specific screening.

[0150] While maintaining a TMP concentration of 20 µg / mL, two rounds (the fourth and fifth rounds) of specific screening for A-to-G editing were conducted to encourage the enzyme to specifically search for key mutations in the evolutionary space that could enhance adenine deamination without limiting cytosine deamination activity.

[0151] 2. Locking of the key site I45V.

[0152] Several candidate variants that showed high enrichment in the screening of two rounds of libraries were selected for activity testing, and the final variant C25-I45V (SEQ ID NO:5, the corresponding expression plasmid is named pMutaT7-C25-I45V) was identified.

[0153] 3. Quantitative characterization of the activity of C25-I45V.

[0154] The mutation rates of each variant determined in parallel are shown in Table 1 below.

[0155] Table 1 The C-to-T editing activity of intermediate C25 was approximately 10-fold (2.32 × 10⁻⁶) higher than that of the starting template (gtTadA-155N-VN). -5 vs 1.98 × 10 -6 However, the increase in A-to-G editing activity was relatively small. After introducing the I45V mutation, the A-to-G editing activity of C25-I45V achieved a leap in improvement (from 2.08 × 10⁻⁶). -6 Upgraded to 2.07 × 10 -5 And it did not negatively affect C-to-T editing activity (maintained at 2.56 × 10⁻⁶). -5 The final variant achieved a mutation rate of 10 in both channels. -5 At a significant scale, a balance of dual-edit activity was successfully achieved.

[0156] Figure 3 This is a multiple sequence alignment diagram of key amino acids in the deaminase variant. This figure shows the amino acid sequence alignment results of the wild type (gtTadA), the truncated intermediate (gtTadA-155N), and the final variant (C25-I45V); it clearly shows multiple key amino acid mutation sites, including I45V, as well as the truncated features of the variant at the C-terminus.

[0157] Figure 4 Characterization of bifunctional editing activity and activity ratios of variants at different evolutionary stages. (A) Mutation frequency of each variant in A-to-G and C-to-T screening quantitative systems ( (B) Editing activity ratio of A-to-G to C-to-T for each variant (dashed line represents the theoretical equilibrium value of 1). As can be seen from the figure, the A-to-G to C-to-T editing activity ratio of variant C25-I45V is closest to 1, showing a highly balanced bidirectional editing capability.

[0158] Example 6 This embodiment provides a fusion protein for genome editing.

[0159] Figure 5This diagram illustrates the structure of a fusion protein for genome editing (using nCas9 as an example). It shows the fusion protein architecture after replacing the localization module with nCas9 (D10A), which includes the NLS nuclear localization signal and the UGI glycosylation inhibitor for precise base editing of the genome.

[0160] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A bifunctional deaminase possessing both adenine and cytosine deamination activities, characterized in that, The amino acid sequence of the bifunctional deaminase includes any of the following: (1) The amino acid sequence as shown in SEQ ID NO:2; (2) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO:2 by substitution, deletion or addition of one or more amino acids and having adenine deamination activity and cytosine deamination activity; (3) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO:2 and has adenine deamination activity and cytosine deamination activity.

2. A bifunctional deaminase mutant with dual deamination activities of adenine and cytosine, characterized in that, The mutant was obtained by mutating the amino acid sequence shown in SEQ ID NO:2; Optionally, the mutation site of the mutant is located at at least one of the following positions in the amino acid sequence shown in SEQ ID NO:2: position 102 or position 104; Optionally, the mutation at position 102 of the mutant is selected from any one of A102V, A102I, or A102L; Optionally, the mutation at position 104 of the mutant is selected from D104N or D104E; Optionally, the mutation site of the mutant is selected from any one or a combination of at least two of A102V, A102I, A102L, D104N or D104E; Optionally, the mutation site is a combination of A102V and D104N; the mutated amino acid sequence is shown in SEQ ID NO:3; Optionally, the mutant is obtained by directed evolution based on the mutant shown in SEQ ID NO:3, and the mutation sites in the directed evolution include any one or a combination of at least two of A13S, E19Q or I56V. Optionally, the mutation sites in directed evolution are a combination of A13S, E19Q and I56V, and the mutated amino acid sequence is shown in SEQ ID NO:4; Optionally, the mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO:4, and the mutation site of the mutant is located at position 45 of the amino acid sequence shown in SEQ ID NO:4; Optionally, the mutation at position 45 of the mutant is selected from any one of I45V, I45L, I45A or I45M; Optionally, the mutation site is I45V, and the mutated amino acid sequence is shown in SEQ ID NO:

5.

3. A nucleic acid base editing fusion protein, characterized in that, The fusion protein includes: (a) The bifunctional deaminase with adenine and cytosine deamination activity as described in claim 1 or the bifunctional deaminase mutant with adenine and cytosine deamination activity as described in claim 2; Optionally, the fusion protein further includes at least one of the following elements: (b) Sequence-specific DNA-binding proteins; (c) Connector to (a) and (b); Optionally, the sequence-specific DNA-binding protein is selected from any one of: T7 RNA polymerase, CRISPR-Cas system effector proteins, or transcription activator-like effector proteins; Optionally, the connector includes at least one of a flexible connector and a rigid connector; Optionally, the CRISPR-Cas system effector protein includes any one of nCas9, dCas9, Cas12a, or Cpf1.

4. A dual-function base editor, characterized in that, The dual-function base editor comprises: the nucleic acid base editing fusion protein of claim 3; Optionally, the bifunctional base editor also includes a guide RNA.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the bifunctional deaminase with adenine and cytosine deamination activity as described in claim 1, a bifunctional deaminase mutant with adenine and cytosine deamination activity as described in claim 2, or the nucleic acid base editing fusion protein as described in claim 3.

6. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule as described in claim 5.

7. A recombinant cell, characterized in that, The recombinant cell contains the expression vector of claim 6, or the genome of the recombinant cell is integrated with the nucleic acid molecule of claim 5.

8. A reagent kit, characterized in that, The kit contains the bifunctional deaminase with adenine and cytosine deamination activity as described in claim 1, the bifunctional deaminase mutant with adenine and cytosine deamination activity as described in claim 2, the nucleic acid base editing fusion protein as described in claim 3, the bifunctional base editor as described in claim 4, or the recombinant cell as described in claim 7.

9. The application of the bifunctional deaminase with adenine and cytosine deamination activities as described in claim 1, the bifunctional deaminase mutant with adenine and cytosine deamination activities as described in claim 2, the nucleic acid base editing fusion protein as described in claim 3, the bifunctional base editor as described in claim 4, the recombinant cells as described in claim 7, or the kit as described in claim 8 in the development of base editing or base editing tools.

10. A method for altering at least one nucleotide in a target DNA sequence, characterized in that, The method includes contacting the target DNA with the bifunctional base editor of claim 4.

11. A screening system, characterized in that, The screening system includes a nucleic acid molecule encoding the nucleic acid base editing fusion protein of claim 3.

12. The screening system according to claim 11, characterized in that, The screening system includes: a bifunctional base editing element containing a nucleic acid molecule encoding the nucleic acid base editing fusion protein of claim 3; Optionally, the screening system further includes a response element; Optionally, the response element includes at least one of a C-to-T response element and an A-to-G response element; Optionally, the screening system is used for quantitative screening and / or evolution of bifunctional deaminases.

13. A method for directed evolution or quantitative activity assessment of bifunctional deaminases using the screening system of claim 11 or 12, characterized in that, The method includes: co-transforming the target deaminase expression element and the response element into host cells; and determining the A-to-G or C-to-T editing activity through resistance screening and / or phenotypic analysis.

14. The method according to claim 13, characterized in that, The directed evolution includes an asymmetric screening step: (1) Provide a mutant library and transform it into a host cell containing the screening system of claim 11 or 12; (2) In the first screening stage, the first editing activity was enriched by adding screening reagents to the culture medium; (3) In the second screening stage, the imbalance of bidirectional activity is corrected by switching the response element for the second editing activity or changing the screening window, and by specifically enriching the second editing activity in the presence of the screening reagent. Optionally, in step (1), the response element includes at least one of a C-to-T response element and an A-to-G response element; Optionally, in step (2), the screening reagent includes trimethoprim; Optionally, the asymmetric screening step further includes alternating between steps (2) and (3) until a bidirectional balanced variant is obtained.

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