Click-to-install genome editing

HK40138114APending Publication Date: 2026-09-25THE GENERAL HOSPITAL CORP
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Patent Information

Application Number
HK62026126433
Authority / Receiving Office
HK · HK
Patent Type
Applications
Current Assignee / Owner
Priority Date
2023-09-08
Filing Date
2026-07-21
Publication Date
2026-09-25
Estimated Expiration
2044-04-07

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Abstract

Here we describe click editing, a series of genome editing approaches that combine DNA nickases (including but not limited to RNA-programmable CRISPR nickases) with DNA ligases or DNA polymerases to perform a range of different genomic edits. The edit of interest is encoded on a nucleic acid template or substrate (optionally a 'click nucleic acid' (clkNA) or 'click DNA' (clkDNA) as described herein), which can be provided in trans or preferentially recruited to the target site via a variety of nucleic acid tethering methods (optionally HUH-family endonucleases (HUHes), Telomere Binding Proteins (TBPs), or DNA- or RNA-binding proteins).
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Description

Abstract This article introduces click editing, a genome editing approach that combines DNA cleavage enzymes (including, but not limited to, RNA-programmable CRISPR cleavage enzymes) with DNA ligases or DNA polymerases to perform a range of different genome edits. The target edit is encoded on a nucleic acid template or substrate (optionally, as described herein, “click nucleic acid” (clkNA) or “click DNA” (clkDNA)), which can be provided trans- or preferentially recruited to the target site binding protein via various nucleic acid ligation methods (optionally, HUH family endonucleases (HUHes), telomere-binding proteins (TBPs), or DNA / RNA-binding proteins).

Claims

Attorney Docket No.29539-0721WO1 / MGH 2023-161 WHAT IS CLAIMED IS:

1. A click editor fusion protein comprising a DNA binding domain (optionally an RNA-programmable DNA nickase or nuclease), a clkNA tethering domain, and an effector domain, with optional linkers therebetween, optionally wherein: (i) the DNA binding domain, clkNA tethering domain, and effector domain are fused in any order; or (ii) the clkNA tethering domain and / or the effector domain is inlaid internally into the DNA binding domain.

2. The click editor fusion proteins of claim 1, wherein the DNA binding domain (optionally an RNA-programmable nickase or nuclease) is a non-target strand nickase or a nuclease (optionally Cas9 nickase or nuclease, Cas12 nickase or nuclease, an IscB nickase or nuclease, a TnpB nickase or nuclease, etc.; see Table C).

3. The click editor fusion proteins of claim 1 or 2, wherein the clkNA tethering domain is: (i) a sequence-specific covalent or non-covalent ssDNA binding domain optionally an HUH endonuclease or telomere binding protein; (ii) a domain that covalently or noncovalently binds a chemical moiety on the clkNA, optionally Avidin, SNAP-tag, CLIP-tag, HALO-tag; or (iii) an RNA-binding domain, optionally an engineered RNA-binding HUH endonuclease or Telomere binding protein, or a Phage coat protein (CP), optionally MCP, PCP, N21p, N22p, BoxB, or Com.

4. The click editor fusion proteins of any of claims 1 to 3, wherein the effector domain is a DNA polymerase or ligase.

5. The click editor fusion proteins of claim 4, wherein the DNA polymerase is a DNA-dependent DNA polymerase of family A, B, C, D, X, or Y, optionally E. coli Klenow (EcKlenow, optionally with the D355A and / or E357A mutations that deactivate its 3’-5’ exonuclease domain); Taq Stoffel; Pol-Beta; Pol-Beta + Sso7d; Phi29 DNA Polymerase (D169A); Sequenase; T4 DNA Polymerase, and E. coli dKlenow (optionally with the D355A, E357A, D705A, and / or D882A mutations), or is a reverse transcriptase, optionally MarathonRT, GsI-IIC RT, and MMLV-RT variants, optionally PE2 MMLV RT (with D200N, T306K, W313F, T330P, L603W mutations) or MMLV or PE2 MMLV RT truncations (optionallyAttorney Docket No.29539-0721WO1 / MGH 2023-161 truncations 2, 5, or 6) or M-MLV RT (T128N, V223Y, D200C, with or without RNaseH domain truncation of M-MLV), HFV RT, HERV RT, LtrA RT, HERV- Kcon RT, Tel4c RT, GsI-IIC RT, Ma-Int5 RT, Gs RT or Gs RT (A16E, L37P, A123V), Tf1 RT or Tf1 RT (P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E, S492N) or Tf1 RT (P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E, S492N, K118R, S188K, I260L, S297Q, R288Q), Ec48 RT or Ec48 RT (E60K, K87E, E165D, D243N, R267I, E279K, K318E, K343N), KORV RT, WMSV RT, AVIRE RT, BAEMV RT, PERV RT, MMTV RT, Ty3 RT, GALV RT, SRV2 RT, MPMV RT, Vp96 RT, ASLV RT, or engineered Marathon (optionally with D14R, N26R, D74R, N116K, or N197R mutations).

6. The click editor fusion proteins of claim 4, wherein the DNA Ligase is T3; T4; T7; ChlV (SplintR); PhiKMV; Vaccinia; or dT4 ligase.

7. A click nucleic acid (clkNA) template, preferably 15-500 nt long, comprising (i) a localization moiety, (ii) a polymerization template (PT) comprising at least one desired edit or an attachment duplex region (ADR) comprising at least one desired edit, and (iii) a flap binding region (FBR), wherein the clkNA template comprises RNA, DNA (clkDNA) or both RNA and DNA.

8. The clkNA template of claim 7, wherein the localization moiety is an HUH endonuclease recognition sequence (RNA or DNA), a Telomere Binding Protein recognition sequence, biotin, benzylguanine derivative, benzylcytosine derivative, a chloroalkane, or an RNA sequence that is bound by an RNA binding protein, optionally a phage coat protein (CP) or phage antitermination signal, optionally MS2, PP7, BoxB, or Com.

9. The clkNA template of claim 7 or 8, wherein the polymerization template (PT) comprises a portion that binds to the target genome, optionally at least 3, 4, 5, 6, 7, 8, 9, or 10 nt long, and up to 500 nt long, and a portion that includes at least one desired edit that is at least 1 nt long.

10. The clkNA template of claim 7 or 8, wherein the ADR comprises a dsDNA portion that comprises a homology to the target genome, optionally at least 3, 4, 5, 6, 7, 8, 9, or 10 nt long, and up to 500 nt long with at least one desired edit that is at least 1 nt long.Attorney Docket No.29539-0721WO1 / MGH 2023-161 11. The clkNA template of any of claims 7 to 10, wherein the flap binding region is complementary to a genomic flap released by a nickase, optionally wherein the flap binding region is 5-50 nt in length, and is immediately 3’ of the PT or ADR.

12. A composition comprising: (i) the click editor fusion protein of any of claims 1-6; (ii) the clkNA template of any of claims 7 to 11, wherein the localization moiety of the clkNA template binds to the clkNA tethering domain of the click editor fusion protein; and (iii) at least one guide RNA that directs the RNA-programmable DNA nickase to a target DNA sequence, and optionally a second guide RNA that directs the RNA- programmable DNA nickase to a target DNA sequence on the opposite strand.

13. A click editor composition comprising a DNA binding domain, a clkNA tethering domain, and an effector domain, optionally wherein the clkNA tethering domain and the DNA binding domain are in a single fusion protein, and the effector domain is separate, and optionally wherein the effector domain is an endogenous DNA-dependent DNA polymerase or endogenous DNA ligase.

14. The click editor composition of claim 13, wherein the effector domain is fused to an RNA binding protein, optionally a phage coat protein (CP), optionally wherein the composition further comprises at least one guide RNA comprising an RNA hairpin sequence that binds the RNA binding protein, optionally the CP.

15. The click editor composition of claim 13, wherein the clkNA tethering domain is fused to the effector domain, and where the DNA binding domain is separate.

16. The click editor composition of claim 13, wherein the clkNA tethering domain is fused to the DNA binding domain on the N terminus or the C terminus, or is inlaid internally into the DNA binding domain.

17. A click editor composition comprising a clkNA tethering domain and a DNA binding domain in a non-covalent complex formed by interaction of protein recruitment domains on each of the clkNA tethering domain and the DNA binding domain, and optionally an effector domain, optionally wherein the effector domain is separate from both the clkNA tethering domain and the DNA binding domain.

18. The click editor composition of claim 17, wherein the protein recruitment domains are interacting coiled coil, leucine zipper, or Suntag-scFv domain pairs.Attorney Docket No.29539-0721WO1 / MGH 2023-161 19. A click editor composition comprising a DNA binding domain, a clkNA tethering domain, and an effector domain, optionally wherein the clkNA tethering domain and the DNA binding domain are in a single fusion protein, and the effector domain is separate.

20. The click editor composition of any of claims 13-19, wherein the DNA binding domain (optionally an RNA-programmable nickase or nuclease) is a non-target strand nickase or a nuclease (optionally Cas9 nickase or nuclease, Cas12 nickase or nuclease, an IscB nickase or nuclease, a TnpB nickase or nuclease, etc.; see Table C).

21. The click editor composition of any of claims 13-20, wherein the clkNA tethering domain is an HUH endonuclease, a Telomere Binding Protein, avidin, SNAP-tag, CLIP-tag, a HALO-tag, or an RNA binding protein, optionally a phage coat protein (CP) or phage antitermination signal, optionally MCP, PCP, Com, N protein; or an engineered RNA-binding HUH endonuclease.

22. The click editor composition of any of claims 13 to 21, wherein the effector domain is a DNA polymerase or ligase.

23. The click editor composition of claim 22, wherein the DNA polymerase is a DNA- dependent DNA polymerase of family A, B, C, D, X, or Y, optionally E. coli Klenow (EcKlenow, optionally with the D355A and / or E357A mutations that deactivate its 3’-5’ exonuclease domain); Taq Stoffel; Pol-Beta; Pol-Beta + Sso7d; Phi29 DNA Polymerase (D169A); Sequenase; T4 DNA Polymerase, and E. coli dKlenow (optionally with the D355A, E357A, D705A, and / or D882A mutations), or is a reverse transcriptase, optionally MarathonRT, GsI-IIC RT, and MMLV-RT variants, optionally PE2 MMLV RT (with D200N, T306K, W313F, T330P, L603W mutations), or MMLV or PE2 MMLV RT truncations (optionally truncations 2, 5, or 6), or M-MLV RT (T128N, V223Y, D200C, with or without RNaseH domain truncation of M-MLV), HFV RT, HERV RT, LtrA RT, HERV- Kcon RT, Tel4c RT, GsI-IIC RT, Ma-Int5 RT, Gs RT or Gs RT (A16E, L37P , A123V ), Tf1 RT or Tf1 RT (P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E, S492N) or Tf1 RT (P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E, S492N, K118R, S188K, I260L, S297Q, R288Q), Ec48 RT or Ec48 RT (E60K, K87E, E165D, D243N, R267I, E279K, K318E, K343N), KORV RT, WMSV RT, AVIRE RT, BAEMV RT,Attorney Docket No.29539-0721WO1 / MGH 2023-161 PERV RT, MMTV RT, Ty3 RT, GALV RT, SRV2 RT, MPMV RT, Vp96 RT, ASLV RT, or engineered Marathon (optionally with D14R, N26R, D74R, N116K, or N197R mutations).

24. The click editor composition of claim 23, wherein the DNA Ligase is T3; T4; T7; ChlV (SplintR); PhiKMV; Vaccinia; or dT4 ligase.

25. The click editor fusion proteins of any of claims 1-6, composition of claim 12, or click editor compositions of any of claims 13-24, further comprising a recombinase fused to the complex or fusion protein comprising the DBD, recruited to the DBD by a protein recruitment domain, or expressed separately in trans.

26. A method of altering a target DNA sequence, optionally a genomic sequence, the method comprising contacting the DNA sequence with: (i) a DNA binding domain (optionally an RNA-programmable DNA nickase or nuclease), a clkNA tethering domain, and an effector domain, optionally in one or more fusion proteins, optionally in the click editor fusion protein of any of claims 1-6, composition of claim 12, or click editor compositions of any of claims 13-25; (ii) the clkNA template of any of claims 7 to 11, wherein the localization moiety of the clkNA template binds to the clkNA tethering domain of the click editor fusion protein; and (iii) a guide RNA that directs the RNA-programmable DNA nickase to the target DNA sequence, wherein: the RNA-programmable DNA nickase nicks the non-target strand at the target site; the FBR on the clkNA template anneals to the non-target DNA strand; and an extended 3’ DNA flap is generated by the effector domain, wherein the 3’ DNA flap is incorporated into the target DNA, leading to altering the target DNA.

27. A method of altering a target DNA sequence, optionally deletion, replacement, or duplication of the target DNA sequence, the method comprising contacting the DNA sequence with: (i) a DNA binding domain (e.g. an RNA-programmable nickase or nuclease), a clkNA tethering domain, and an effector domain, optionally in one or more fusion proteins, optionally in the click editor fusion protein of any of claims 1-6,Attorney Docket No.29539-0721WO1 / MGH 2023-161 composition of claim 12, or click editor compositions of any of claims 13-25; (ii) the clkNA template of any of claims 7 to 11, wherein the localization moiety of the clkNA template binds to the clkNA tethering domain of the click editor fusion protein; and (iii) a pair of sgRNAs, each targeting opposite DNA strands, to generate two extended 3’ flaps that are either complementary to upstream sequence (relative to the nick site) or to each other (and sufficiently dissimilar to the DNA between the two nicks).

28. A method of altering a target DNA sequence, optionally a genomic sequence, the method comprising contacting the DNA sequence with: (i) a DNA binding domain (e.g. an RNA-programmable DNA nickase or nuclease) linked to a clkNA tethering domain with optional linkers therebetween, (ii) the clkNA template of any of claims 7 to 11, wherein the localization moiety of the clkNA template binds to the clkNA tethering domain; (iii) an effector domain linked to an RNA binding protein, optionally MCP, PCP, or Com RNA binding protein; and (iv) one or a pair of sgRNAs, each targeting opposite DNA strands, to generate two extended 3’ flaps that are either complementary to upstream sequence (relative to the nick site) or to each other (and sufficiently dissimilar to the DNA between the two nicks), wherein one or both of the sgRNAs comprises a MS2, PP7, or com sequence.

29. A method of altering a target DNA sequence, optionally a genomic sequence, the method comprising contacting the DNA sequence with: (i) a DNA binding domain (optionally an RNA-programmable DNA nickase or nuclease), a clkNA tethering domain, and an effector domain, optionally in one or more fusion proteins, optionally in the click editor fusion protein of any of claims 1-6, composition of claim 12, or click editor compositions of any of claims 13-25; (ii) the clkNA template of any of claims 7 to 11, comprising a localization moiety, a polymerization template (PT) comprising at least one desired edit or an attachment duplex region (ADR) comprising at least one desired edit, a first flap binding region (PBS1), and the reverse complement of a second PBS (rcPBS 2), wherein the localization moiety of the clkNA template binds to the clkNAAttorney Docket No.29539-0721WO1 / MGH 2023-161 tethering domain of the click editor fusion protein; and (iii) a pair of guide RNAs that direct the RNA-programmable DNA nickase to first and second sites on the target DNA sequence, wherein: the RNA-programmable DNA nickase creates first and second nicks on the non- target strand at the target site; the PBS1 on the clkNA template anneals to the non-target DNA strand at the first nick site; an extended 3’ DNA flap comprising a sequence complementary to PBS2 is generated by the effector domain, the extended 3’ DNA flap comprising PBS2 anneals to the 3’ flap at the second nick site; the effector domain carries out second strand synthesis; and the newly synthesized DNA is incorporated into the target DNA, leading to altering the target DNA.

30. The method of any of claims 26-29, wherein the edit comprises insertion of an attP or attB sequence, and the method further comprises contacting the DNA with a donor template comprising attR and attL sequences, and a serine recombinase, optionally BxBl or Pa01, optionally fused to the click editor fusion protein.

31. The clkNA template of any of claims 7-11 or composition of claim 12, wherein the edit comprises insertion of an attP or attB sequence.

32. The clkNA template or composition of any of claims 7-12 or 31, wherein the clkNA template is all DNA (clkNA) or partly DNA and partly RNA (e.g. the HUH endonuclease sequence is DNA and the rest is RNA; the HUH endonuclease sequence is DNA, all or a portion of the FBR is RNA and the PT is DNA; or the HUH endonuclease sequence is DNA, and all or a portion of the FBR is RNA and all or a portion of the PT is RNA; or any of the above where the HUH endonuclease sequence is RNA instead of DNA).

33. The clkNA template or composition of any of claims 7-12, 30, 31, or 32, wherein the clkNA template comprises one or more chemical modifications, optionally a modified sugar moiety and / or a modified internucleoside linkage.