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CN-122012476-A - Difunctional deaminase with adenine and cytosine bidirectional deamination activity, mutant thereof, screening method and application

CN122012476ACN 122012476 ACN122012476 ACN 122012476ACN-122012476-A

Abstract

The invention provides a difunctional deaminase with adenine and cytosine bidirectional deamination activity, mutants thereof, a screening method and application. The amino acid sequence of the bifunctional deaminase comprises any one of (1) an amino acid sequence shown as SEQ ID NO. 2, (2) an amino acid sequence which is derived from the amino acid sequence shown as 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 shown as SEQ ID NO. 2 and has adenine deamination activity and cytosine deamination activity. The single domain bifunctional deaminase variant with high activity and balanced bidirectional deamination capacity is obtained through directed evolution, and the variant remarkably improves editing activity, and can realize bidirectional editing and balanced bidirectional deamination capacity only through a single polypeptide chain.

Inventors

  • ZHANG TINGKAI
  • TANG JINLE
  • ZHANG ZHE
  • ZHAN JIAN
  • ZHOU YAOQI

Assignees

  • 深圳湾实验室

Dates

Publication Date
20260512
Application Date
20260224
Priority Date
20260129

Claims (14)

  1. 1. A bifunctional deaminase having adenine and cytosine bidirectional deamination activity, wherein the amino acid sequence of the bifunctional deaminase comprises any one of the following: (1) An amino acid sequence shown as SEQ ID NO. 2; (2) An amino acid sequence which is derived from the amino acid sequence shown in SEQ ID NO.2 by substitution, deletion or addition of one or more amino acids and has adenine deamination activity and cytosine deamination activity; (3) An amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO. 2 and having adenine deamination activity and cytosine deamination activity.
  2. 2. A difunctional deaminase mutant with adenine and cytosine bidirectional deamination activity is characterized in that the mutant is obtained by mutation on the basis of an amino acid sequence shown in SEQ ID NO. 2; Optionally, the mutation site of the mutant is located at least one of the following positions 102 or 104 of the amino acid sequence shown in SEQ ID NO. 2; Optionally, the mutation at position 102 of the mutant is selected from any one of a102V, A102I or a 102L; Optionally, the mutation at position 104 of the mutant is selected from D104N or D104E; optionally, the mutant mutation site is selected from any one or a combination of at least two of a102V, A102I, A102L, D N or D104E; Optionally, the mutation site is a combination of A102V and D104N, and the amino acid sequence after mutation is shown as SEQ ID NO. 3; optionally, the mutant is obtained after directed evolution based on the mutant shown in SEQ ID NO. 3, and the mutation site in the directed evolution comprises any one or a combination of at least two of A13S, E Q or I56V; Optionally, the mutation site in directed evolution is a combination of A13S, E Q and I56V, and the amino acid sequence after mutation is shown as SEQ ID NO. 4; Optionally, the mutant is obtained by mutation based on the amino acid sequence shown in SEQ ID NO. 4, and the mutation site of the mutant is positioned at the 45 th position 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, I a or I45M; optionally, the mutation site is I45V, and the amino acid sequence after mutation is shown as SEQ ID NO. 5.
  3. 3. A nucleobase editing fusion protein, said fusion protein comprising: (a) The bifunctional deaminase having adenine and cytosine bidirectional deamination activity of claim 1 or the bifunctional deaminase mutant having adenine and cytosine bidirectional deamination activity of claim 2; optionally, the fusion protein further comprises at least one of the following elements: (b) A sequence-specific DNA binding protein; (c) A linker connecting (a) and (b); Optionally, the sequence-specific DNA binding protein is selected from any one of T7 RNA polymerase, CRISPR-Cas system effector protein or transcription activator-like effector protein; optionally, the connector comprises at least one of a flexible connector and a rigid connector; optionally, the CRISPR-Cas system effector protein comprises any one of nCas, dCAS9, cas12a or Cpf 1.
  4. 4. A dual function base editor, the dual function base editor comprising: the nucleobase editing fusion protein of claim 3; Optionally, the bifunctional base editor further comprises a guide RNA.
  5. 5. A nucleic acid molecule encoding the bifunctional deaminase with adenine and cytosine bidirectional deamination activity of claim 1, encoding the bifunctional deaminase mutant with adenine and cytosine bidirectional deamination activity of claim 2 or encoding the nucleobase editing fusion protein of claim 3.
  6. 6. An expression vector comprising the nucleic acid molecule of claim 5.
  7. 7. A recombinant cell comprising the expression vector of claim 6 or having incorporated into its genome the nucleic acid molecule of claim 5.
  8. 8. A kit comprising the bifunctional deaminase having adenine and cytosine bidirectional deamination activity of claim 1, the bifunctional deaminase mutant having adenine and cytosine bidirectional deamination activity of claim 2, the nucleobase editing fusion protein of claim 3, the bifunctional base editor of claim 4 or the recombinant cell of claim 7.
  9. 9. Use of the bifunctional deaminase with adenine and cytosine bidirectional deamination activity of claim 1, the bifunctional deaminase mutant with adenine and cytosine bidirectional deamination of claim 2, the nucleobase editing fusion protein of claim 3, the bifunctional base editor of claim 4, the recombinant cell of claim 7 or the kit of claim 8 in base editing or development of a base editing tool.
  10. 10. A method of altering at least one nucleotide in a target DNA sequence, comprising contacting the target DNA with the bifunctional base editor of claim 4.
  11. 11. A screening system comprising a nucleic acid molecule encoding the nucleobase editing fusion protein of claim 3.
  12. 12. The screening system of claim 11, comprising a bifunctional base editing element comprising a nucleic acid molecule encoding the nucleic acid base editing fusion protein of claim 3; Optionally, the screening system further comprises a response element; optionally, the response element comprises at least one of a C-to-T response element, an A-to-G response element; optionally, the screening system is used for quantitative screening and/or evolution of bifunctional deaminase.
  13. 13. A method for directed evolution or quantitative assessment of activity of bifunctional deaminase using the screening system of claim 11 or 12, characterized in that the method comprises co-transforming the deaminase expression element to be tested with the response element into host cells and determining the editing activity of A-to-G or C-to-T by resistance screening and/or phenotyping.
  14. 14. The method of claim 13, wherein the directed evolution comprises an asymmetric screening step: (1) Providing a library of mutations and transforming it into host cells comprising the screening system of claim 11 or 12; (2) Enrichment for the first editing activity is performed in a first screening stage by adding a screening reagent to the medium; (3) In a second screening stage, correcting the imbalance of bidirectional activity by switching response elements or changing screening windows for a second editing activity and performing specific enrichment for the second editing activity in the presence of a screening agent; optionally, in step (1), the responsive element comprises at least one of a C-to-T responsive element, an a-to-G responsive element; Optionally, in step (2), the screening agent comprises trimethoprim; Optionally, the asymmetric screening step further comprises alternating steps (2) and (3) above until a bi-directional equalized variant is obtained.

Description

Difunctional deaminase with adenine and cytosine bidirectional deamination activity, mutant thereof, screening method and application The application claims priority from 2026101285400 patent application (the application date of the prior application is 2026, 1 and 29 days), and the application is a bifunctional deaminase with adenine and cytosine bidirectional deamination activity, and mutants and application thereof. Technical Field The invention belongs to the field of biotechnology and gene editing, and particularly relates to a bifunctional deaminase with adenine and cytosine bidirectional deamination activity, a mutant thereof, a screening method and application thereof. Background Accurate genome editing is a core technology for analyzing gene functions, treating genetic diseases and performing genetic improvement on crops. Early gene editing tools, such as meganucleases, zinc Finger Nucleases (ZFNs) and transcription activation-like effector nucleases (TALENs), achieved cleavage of specific DNA sequences by protein-DNA specific recognition, but because of the reliance on protein engineering redirection, were complex in design, high in construction cost and difficult to achieve high-throughput applications. The CRISPR-Cas system is found, especially the application of Cas9 nuclease, and the targeting of specific sites of the genome is realized through a simple RNA-DNA base complementary pairing principle, so that the gene editing field is thoroughly innovated. Traditional CRISPR-Cas editing typically relies on introducing DNA Double Strand Breaks (DSBs) at the target site followed by cell endogenous non-homologous end joining (NHEJ) or homologous recombination repair (HDR) pathways (1) NHEJ, while efficient, often produces uncontrolled insertions/deletions (indels) that easily result in frame shifts and loss of function in the open reading frame, (2) HDR relies on homologous templates, which are less efficient and active mainly in dividing cells, and are difficult to fully function in many application scenarios, (3) excessive DSBs can also induce genomic rearrangements, large fragment deletions, and p 53-mediated DNA damage responses, with potential safety risks. Therefore, the development of DSB-independent precise base rewriting technology has become an important direction in the field of gene editing. To overcome the problems with DSBs, researchers have developed a Base Editor (BE) that enables chemical conversion of specific bases without cleaving double-stranded DNA by fusing an inactivated or partially inactivated Cas protein with a base deaminase. Cytosine Base Editor (CBE) in which a CBE is based on cytosine deaminase (e.g. rAPOBEC A, A3A) to deaminate cytosine (C) to uracil (U) and read as thymine (T) during replication or repairIs a base transition of (a). A typical system such as BE3/BE4 obtains higher efficiency through APOBEC 1-nCas-UGI combination, but side C editing, C.fwdarw.A/C.fwdarw.G transversions and a certain proportion of insertions/deletions (indels) are easy to occur. Adenine deaminase which uses DNA as a substrate is lacking in nature. The existing research obtains the DNA which can act on single strand through the directed evolution of E. coli TadA (hereinafter abbreviated as ecTadA)And fusion with nCas to construct ABE7.10 to realizeAnd then series of tools such as ABE8e, ABE9 and the like are developed, and the editing efficiency and compatibility are further improved. A dual-function base editor, wherein a single CBE or ABE can only catalyze one type of base conversion, and has limitation when a complex mutation site of A and C needs to be rewritten simultaneously or a full-coverage saturated mutation library is constructed in the same target region. Therefore, the prior work has developed double-function base editors such as A & C-BEmax, target-ACE/ACEmax and SPACE by connecting APOBEC family cytosine deaminase and ABE7.10/ABE8e to the same nCas protein in series, and realizes the synchronous editing of C-to-T and A-to-G at the same Target site. However, there are several common problems with such a dual-function editor with two deaminase enzymes in tandem: (1) The protein has large volume and limited delivery, and the fusion protein formed by one Cas protein and two deaminase domains has larger molecular weight, is not beneficial to loading into adeno-associated virus and other capacity-limited vectors, and increases the expression burden of a host. (2) The dual-function editing activity is difficult to balance, namely, the substrate ranges, editing windows and optimal working conditions of different deaminase are different, the situation that one type of editing is obviously stronger and the other type of editing is relatively weaker often occurs, and the balanced A/C editing in the same window is difficult to obtain. In this context, it is of great importance to develop a novel deaminase chassis based on homologs of different species sources TadA, in particular an en