CRISPR enzymes and systems with modified pam specificity

Patent metadata

Field	Value
Publication number	US-12410416-B2
Application number	US-201716095170-A
Country	US
Kind code	B2
Filing date	Apr 19, 2017
Priority date	Apr 19, 2016
Publication date	Sep 9, 2025
Grant date	Sep 9, 2025

Abstract

The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA-targeting systems comprising a novel DNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA. Aspects of the invention in particular relate to Cpf1 mutants having altered PAM specificity.

First claim

What is claimed:

1. A mutated Cpf1 polypeptide comprising one or more mutations affecting protospacer-adjacent motif (PAM) recognition, wherein the mutated Cpf1 polypeptide is a non-naturally-occurring Acidaminococcus sp. Cpf1 (AsCpf1) or orthologue of AsCpf1, wherein the mutated Cpf1 polypeptide recognizes a PAM sequence which is not recognized by wild type AsCpf1 or orthologue of AsCpf1, and wherein the mutated Cpf1 polypeptide has at least 95% sequence identity with wild type AsCpf1 or orthologue of AsCpf1,
wherein the mutated Cpf1 polypeptide is
(i) a mutated AsCpf1 comprising one of the following combinations of mutations: S542R and K607R, or S542R and K548V, or S542R and K548R, or S542R, K548V and N552R of AsCpf1 compared to wild type AsCpf1 having the sequence as set forth in SEQ ID NO: 359; or
(ii) a mutated Lachnospiraceae bacterium (LbCpf1) comprising one of the following combinations of mutations: G532R and K595R; G532R and K538V; or G532R, K538V and Y542R of LbCpf1 compared to wild type LbCpf1 having the sequence as set forth in SEQ ID NO: 360, or
(iii) a mutated Francisella tularensis subsp. Novicida Cpf1 (FnCpf1) polypeptide comprising a combination of mutations corresponding to one of the following combinations of mutations of AsCpf1: S542R and K607R, or S542R and K548V, or S542R and K548R, or S542R, K548V and N552R of AsCpf1 compared to wild type AsCpf1 having the sequence as set forth in SEQ ID NO: 359; or
(iv) a mutated Moraxella bovoculi 237 Cpf1 (MbCpf1) polypeptide comprising a combination of mutations corresponding to one of the following combinations of mutations of AsCpf1: S542R and K607R, or S542R and K548V, or S542R and K548R, or S542R, K548V and N552R of AsCp1I compared to wild type AsCpf1 having the sequence as set forth in SEQ ID NO: 359.

2. The mutated Cpf1 polypeptide according to claim 1 , wherein said mutated Cpf1 is not mutated FnCpf1.

3. The mutated Cpf1 polypeptide according to claim 1 , wherein the mutated Cpf1 recognizes a PAM having a sequence YCN, (T)YCV, AYV, TYV, RYN, or RCN or having a sequence TNYC or TNYS, wherein said PAM is not TTTV, and/or said PAM is not TTTC, and/or said PAM is not TCTS.

4. The mutated Cpf1 polypeptide according to claim 3 , wherein said PAM has a sequence or comprises a sequence (T)YCV or RYN; AYV or TYV; YCN or RCN.

5. The mutated Cpf1 polypeptide according to The mutated Cpf1 polypeptide according to wherein the mutated Cpf1 recognizes a PAM having a sequence or comprising a sequence YCN, YCV, AYV, TYV, RYN, RCN, TGYV, NTTN, TTN, TRTN, TYTV, TYCT, TYCN, TRTN, TACT, TYCC, TRTC, TATV, NTTV, TTV, TSTG, TVTS, TYYS, TCYS, TBYS, TCYS, TNYS, TYYS, TNTN, TSTG, TTCC, TCCC, TATC, TGTG, TCTG, TYCV, or TCTC.

6. The mutated Cpf1 polypeptide according to claim 1 , wherein said mutated Cpf1 is mutated AsCpf1 or mutated LbCpf1.

7. The mutated Cpf1 polypeptide according to claim 1 , further comprising modifications or mutations affecting Cpf1 catalytic activity and/or Cpf1 stability.

8. The mutated Cpf1 polypeptide according to claim 1 , which is comprised in a fusion protein with a functional domain.

9. The mutated Cpf1 polypeptide according to claim 8 , wherein said functional domain comprises an activator domain, a repressor domain, a recombinase, a transposase, a histone remodeler, a DNA methyltransferase, a cryptochrome, a light inducible/controllable domain, or a chemically inducible/controllable domain.

10. The mutated Cpf1 polypeptide according to claim 1 which is a nickase.

11. The mutated Cpf1 polypeptide according to claim 1 which is a catalytically inactive Cpf1 polypeptide.

12. The mutated Cpf1 polypeptide according to claim 1 , wherein the Cpf1 polypeptide has at least 95% identity to the sequence of SEQ ID NO: 359, and
wherein the Cpf1 polypeptide recognizes a PAM other than TTTC.

13. The mutated Cpf1 polypeptide according to claim 1 , wherein the Cpf1 polypeptide has at least 95% identity to the sequence of SEQ ID NO: 360,
wherein the one or more mutations are selected from the group consisting of G532R/K595R and G532R/K538V/Y542R of LbCpf1 of SEQ ID NO: 360, and
wherein the Cpf1 polypeptide recognizes a PAM other than TTTC.

14. An isolated host cell comprising the mutated Cpf1 polypeptide according to claim 1 .

15. A method of modifying or targeting a target DNA locus, the method comprising delivering to said locus the mutated Cpf1 polypeptide according to claim 1 and a guide RNA, wherein said mutated Cpf1 polypeptide forms a complex with said guide RNA and wherein said target DNA locus is modified or targeted upon binding of said complex to said target DNA locus.

16. An engineered, non-naturally occurring Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising
a) a guide RNA which comprises a guide sequence linked to a direct repeat sequence, wherein the guide sequence is capable of hybridizing with a target sequence adjacent to a Protospacer Adjacent Motif (PAM), or one or more nucleotide sequences encoding the guide RNA, and
b) the mutated Cpf1 polypeptide of claim 1 ;
wherein said guide RNA is capable of forming a complex with the mutated Cpf1 polypeptide.

17. A polynucleotide encoding a mutated Cpf1 polypeptide comprising one or more mutations affecting protospacer-adjacent motif (PAM) recognition, wherein said mutated Cpf1 polypeptide is a non-naturally-occurring Acidaminococcus sp. Cpf1 (AsCpf1) or orthologue of AsCpf1, wherein the mutated Cpf1 polypeptide recognizes a PAM sequence which is not recognized by the wild type AsCpf1 or orthologue of AsCpf1, and wherein the mutated Cpf1 polypeptide has at least 95% sequence identity with wild type AsCpf1 or orthologue of AsCp1,
wherein the mutated Cpf1 polypeptide is
(i) a mutated AsCpf1 comprising one of the following combinations of mutations: S542R and K607R, or S542R and K548V, or S542R and K548R, or S542R, K548V and N552R of AsCpf1 compared to wild type AsCpf1 having the sequence as set forth in SEQ ID NO: 359; or
(ii) a mutated Lachnospiraceae bacterium (LbCpf1) comprising one of the following combinations of mutations: G532R and K595R; G532R and K538V; or G532R, K538V and Y542R of LbCp1 compared to wild type LbCp1 having the sequence as set forth in SEQ ID NO: 360, or
(iii) a mutated Francisella tularensis subsp. Novicida Cpf1 (FnCpf1) polypeptide comprising a combination of mutations corresponding to one of the following combinations of mutations of AsCpf1: S542R and K607R, or S542R and K548V, or S542R and K548R, or S542R, K548V and N552R of AsCpf1 compared to wild type AsCpf1 having the sequence as set forth in SEQ ID NO: 359; or
(iv) a mutated Moraxella bovoculi 237 Cpf1 (MbCpf1l polypeptide comprising a combination of mutations corresponding to one of the following combinations of mutations of AsCpf1: S542R and K607R, or S542R and K548V, or S542R and K548R, or S542R, K548V and N552R of AsCpf1 compared to wild type AsCpf1 having the sequence as set forth in SEQ ID NO: 359.

18. The polynucleotide according to claim 17 , which is codon optimized for expression in a cell of interest.

19. The polynucleotide according to claim 17 , wherein said polynucleotide further comprises one or more sequence encoding one or more nuclear localization signal (NLS).

20. A vector comprising the polynucleotide according to claim 17 .

21. An engineered, non-naturally occurring Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) vector system

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology

Inventors

• Zhang Feng
• Zetsche Bernd
• Gao Linyi
• Slaymaker Ian
• Cox David Benjamin Turitz

Classifications

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• C12N15/907

  in mammalian cells · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title