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CN-122029275-A - Conditional DNA modifying enzymes comprising heterologous DNA binding domains

CN122029275ACN 122029275 ACN122029275 ACN 122029275ACN-122029275-A

Abstract

The present invention provides a method of identifying amino acid positions of a DNA modification enzyme for insertion into a heterologous DNA Binding Domain (DBD), the method comprising the steps of providing a library of DNA modification enzymes, wherein members of the library comprise heterologous amino acid sequence insertions of the entire DNA modification enzyme, identifying those DNA modification enzymes in the library that have DNA modification activity, and determining the insertion positions in those DNA modification enzymes identified in the previous step. The invention also relates to methods of making DNA modifying enzymes comprising inserted heterologous DBDs, methods of modifying nucleic acid sequences in cells, and methods of evolving DNA binding domains for desired target sequences. The invention also provides nucleic acid sequences encoding such DNA modifying enzymes and DNA binding domains, corresponding vectors and host cells.

Inventors

  • 50. Muhammechianova
  • F. Buchholtz
  • J. Torres Rivera

Assignees

  • 德累斯顿工业大学公法团体

Dates

Publication Date
20260512
Application Date
20241016
Priority Date
20231016

Claims (15)

  1. 1. A method of identifying an amino acid position of a DNA modifying enzyme for insertion into a heterologous DNA Binding Domain (DBD), the method comprising the steps of: (i) Providing a library of DNA modifying enzymes, wherein members of the library comprise heterologous amino acid sequence insertions of the entire DNA modifying enzyme; (ii) Identifying those DNA modifying enzymes in the library having DNA modifying activity, and (Iii) Determining the insertion position in those DNA modifying enzymes identified in step (ii).
  2. 2. The method according to claim 1, wherein the one or more insertion positions determined in step (iii) are mapped to structural data of the DNA modifying enzyme, and/or Wherein the library of DNA modification enzymes provided in (i) is encoded by a nucleic acid library, preferably wherein step (iii) comprises determining at least a part of the nucleic acid sequences encoding those DNA modification enzymes identified in step (ii), and/or The method further comprises the step of selecting one or more amino acid positions for insertion into a heterologous DBD that is exposed to the surface in the DNA modifying enzyme and is proximal to the DNA binding site of the DNA modifying enzyme, and/or Wherein each member of the library of DNA modifying enzymes independently of the other comprises a heterologous amino acid sequence of 3 to 10 amino acids in length, preferably 5 amino acids in length.
  3. 3. A method of producing a DNA modifying enzyme comprising a heterologous DBD insertion, the method comprising the steps of: (i) Inserting a nucleic acid sequence encoding a heterologous DBD into a nucleic acid sequence encoding a DNA modifying enzyme at a nucleotide triplet encoding one or more positions identified in the method of claim 1 or 2, and (Ii) Expressing the nucleic acid sequence produced in step (i).
  4. 4. The method according to claim 3, wherein the nucleic acid sequence encoding a heterologous DBD further comprises a nucleic acid sequence encoding a peptide linker upstream and downstream of the nucleic acid sequence encoding a heterologous DBD, preferably wherein the linker is a glycine-serine linker, preferably a glycine-serine linker with at least one G to R substitution.
  5. 5. The method according to any one of claims 1-4, wherein the DBD is a Zinc Finger (ZF) DBD or a transcription activator-like effector (TALE) DBD, and/or wherein the DNA modifying enzyme is a transposase or a recombinase, preferably a serine recombinase or a tyrosine recombinase, more preferably a tyrosine recombinase.
  6. 6. A DNA modification enzyme comprising a heterologous DBD insertion, Obtained by a process according to any one of claims 3 to 5, and/or Wherein the method comprises the steps of (I) The DNA modification enzyme is Cre or Cre-derived recombinase and the DBD is inserted between amino acid positions 278 and 279, or (Ii) The DNA modification enzyme is Vika or Vika-derived recombinase, and the DBD is inserted between amino acid positions 172 and 173, or (Iii) The DNA modification enzyme comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs 5, 9-13, 19, 20, 22, 30, 41, 42, 51, 64 and 91-200.
  7. 7. A DNA modifying enzyme comprising a heterologous DBD insertion, wherein the DBD optionally comprises a peptide linker at its N-and/or C-terminus, wherein the DNA modifying enzyme is inactive to its target site when the heterologous DBD is not bound to its target DNA, and wherein the DNA modifying enzyme is active to its target site when the heterologous DBD is bound to its target DNA.
  8. 8. A nucleic acid or nucleic acids encoding a DNA modifying enzyme according to claim 6 or 7.
  9. 9. An expression vector comprising a nucleic acid or a plurality of nucleic acids according to claim 8.
  10. 10. A host cell or host cell culture comprising a nucleic acid or a plurality of nucleic acids according to claim 8, or an expression vector according to claim 9, preferably wherein the host cell expresses a DNA modifying enzyme encoded by the nucleic acid or nucleic acids.
  11. 11. A pharmaceutical composition comprising a DNA modifying enzyme according to any one of claims 6 or 7, a nucleic acid or nucleic acids according to claim 8, an expression vector according to claim 9 or a host cell or host cell culture according to claim 10, and a pharmaceutically acceptable excipient or carrier.
  12. 12. Use of a DNA modifying enzyme according to any one of claims 6 or 7, a nucleic acid or nucleic acids according to claim 8, an expression vector according to claim 9, a host cell or host cell culture according to claim 10 or a pharmaceutical composition according to claim 11 for modifying a nucleic acid sequence of interest.
  13. 13. A method of modifying a nucleic acid sequence of interest, the method comprising contacting a cell or tissue comprising the nucleic acid sequence of interest with a DNA modifying enzyme according to any one of claims 6 or 7, a nucleic acid or nucleic acids according to claim 8, an expression vector according to claim 9, a host cell or host cell culture according to claim 10, or a pharmaceutical composition according to claim 11, under conditions that allow the DNA modifying enzyme to modify the nucleic acid sequence of interest.
  14. 14. A method of altering the specificity and/or activity of a DNA modifying enzyme, the method comprising the steps of: (i) The method according to claim 1 or 2, identifying the amino acid position of a DNA modifying enzyme for insertion into a heterologous DBD, and (Ii) Inserting a heterologous DBD at the position identified in step (i).
  15. 15. A method of evolving a DBD for a target sequence of interest, the method comprising the steps of: (i) Creating a library of DBD variants; (ii) Cloning the library of step (i) into an expression vector comprising a first region encoding a DNA recombinase, a first target site comprising the DNA recombinase and a second region flanking the first target site, and a second target site comprising the DNA recombinase and a third region flanking the second target site, such that a DBD is inserted into the DNA recombinase directly or through a peptide linker, wherein the first, second and third regions are separate from each other; (iii) Introducing the expression vector into a host cell and culturing the host cell, thereby expressing the encoded DNA recombinase comprising DBD; (iv) Isolating plasmids from the cell culture of step (iii) and determining whether the DNA recombinase catalyzes a recombination reaction at two target sites on the vector; (v) Amplifying DBDs of those plasmids found to encode DNA recombinases comprising DBDs and exhibiting recombinant activity using error-prone PCR to generate a new library of DBD variants; (vi) Repeating steps (ii) - (iv) with the library of step (v).

Description

Conditional DNA modifying enzymes comprising heterologous DNA binding domains Technical Field The present invention relates to DNA modifying enzymes having conditional activity conferred by insertion of a heterologous DNA binding domain. The invention also relates to methods of producing such DNA modifying enzymes, methods of modifying nucleic acid sequences in cells, and methods of evolving DNA binding domains for desired target sequences. The invention also provides nucleic acid sequences encoding such DNA modifying enzymes and DNA binding domains, corresponding vectors and host cells. Background Tyrosine site-specific recombinases (Y-SSR), such as the Cre-loxP system, are widely used genome editing tools with tremendous therapeutic application potential due to their precise DNA manipulation mechanism. Y-SSR can perform complex genomic engineering operations, including excision, inversion, integration and cassette exchange of large genomic sequences, without inducing DNA double strand breaks. They are also independent of cellular DNA repair mechanisms or other cofactors. Thus, the DNA editing process is predictable and effective even in non-dividing cells (reviewed in Meinke et al, 2016). Despite these unique and useful properties, the laborious stepwise directed molecular evolution and protein engineering required to reprogram it to the target-defined locus (Buchholz&Stewart, 2001;Sarkar et al., 2007;Buchholz&Hauber, 2011;Karpinski et al., 2016;Lansing et al., 2020;Lansing et al., 2022;Rojo-Romanos et al., 2023). has limited the popularization of recombinant enzymes as a multifunctional genome editing tool. On the other hand, programmable nucleases, such as ZFNs, TALENs and CRISPR-systems, can be easily programmed and adapted to different applications, which lays the foundation for the genome editing revolution (Caroll, 2017;Anzalone et al, 2020;Wang&Doudna 2023). However, the introduction of nucleases into DNA gaps or double strand breaks, which require a subsequent repair process by the host cell, often results in undesirable side effects (Kosicki et al., 2018;Adikusuma et al., 2018;Enache et al., 2020;Leibowitz et al., 2021;Papathanasiou et al., 2021;Sinha et al., 2021). combining the accuracy of the recombinase with the convenience of targeting the programmable nucleases, is expected to provide the most useful genome editing tool in the industry. It is therefore an object of the present invention to provide DNA modifying enzymes with conditionally active and/or altered specificity. Disclosure of Invention The object of the present invention is solved by providing a method for identifying the amino acid position of a DNA modifying enzyme for insertion into a heterologous DNA Binding Domain (DBD), said method comprising the steps of: (i) Providing a library of DNA modifying enzymes, wherein members of the library comprise heterologous amino acid sequence insertions of the entire DNA modifying enzyme; (ii) Identifying those DNA modifying enzymes in the library having DNA modifying activity, and (Iii) Determining the insertion position in those DNA modifying enzymes identified in step (ii). According to one embodiment, one or more of the insertion positions determined in step (iii) are mapped to structural data of the DNA modifying enzyme. According to another embodiment, the library of DNA modifying enzymes provided in step (i) is encoded by a nucleic acid library. According to another embodiment, step (iii) comprises determining at least a portion of the nucleic acid sequence encoding those DNA modifying enzymes identified in step (ii). According to one embodiment, the method further comprises the step of selecting one or more amino acid positions for insertion of a heterologous DBD, which is exposed to the surface in the DNA modifying enzyme and is proximal to the DNA binding site of the DNA modifying enzyme. According to another embodiment, the heterologous amino acid sequences comprised in each member of the library of DNA modifying enzymes independently of each other are 3 to 10 amino acids in length, preferably 5 amino acids in length. According to another aspect, the present invention provides a method of producing a DNA modifying enzyme comprising heterologous DBD insertion, the method comprising the steps of: (i) Inserting a nucleic acid sequence encoding a heterologous DBD into a nucleic acid sequence encoding a DNA modifying enzyme at a nucleotide triplet encoding one or more positions identified in the method of claims 1-6, and (Ii) Expressing the nucleic acid sequence produced in step (i). According to one embodiment, the nucleic acid sequence encoding the heterologous DBD further comprises a nucleic acid sequence encoding a peptide linker upstream and downstream of the nucleic acid sequence encoding the heterologous DBD. Preferably, the linker is a glycine-serine linker, more preferably a glycine-serine linker having at least one G to R substitution. According to a preferred embodiment, th