IP Library › Granted Patent US 12,590,299
Granted Patent B2
US 12,590,299 · App. 17/450,593 · Granted Mar 31, 2026

Variants of CRISPR from

Inventors: J. Keith Joung (Winchester, MA); Benjamin Kleinstiver (Boston, MA)
Assignee: The General Hospital Corporation
C12N9/22C07K14/39C12N9/0071C12N15/11C12N15/907C12Y114/11C12Y301/21004C07K2319/00C07K2319/80C12N2310/20
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Quick Facts
Patent No.
US 12,590,299
App. No.
17/450,593
Granted
Mar 31, 2026
Kind
B2
Abstract

Engineered CRISPR from Prevotella and Francisella 1 (Cpf1) nucleases with altered and improved target specificity and their use in genomic engineering, epigenomic engineering, genome targeting, genome editing, and in vitro diagnostics.

Claims (33)

1 . An isolated CRISPR from Prevotella and Francisella 1 (Cpf1) protein, wherein the protein is from Lachnospiraceae bacterium ND2006 (LbCpf1), comprising a sequence that is at least 80% identical to the amino acid sequence of amino acids 1-1228 of SEQ ID NO: 10, with mutations at one or more of the following positions: N160, S168, N256, N260, K272, S286, K349, D505, Q513, G741, Q944, K945, and/or S985 of amino acids 1-1228 of SEQ ID NO:10.

2 . The isolated protein of claim 1 , comprising one or more of the following mutations: N160A, S168A, N256A, N260A, K272A, S286A, K349A, D505A, Q513A, G741A, Q944A, K945A, and/or S985A.

3 . The isolated protein of claim 1 , further comprising one or more mutations that decrease nuclease activity selected from the group consisting of mutations at D832 and E925.

4 . The isolated protein of claim 3 , comprising mutations D832A and E925A.

5 . The isolated protein of claim 1 , comprising a sequence that is at least 95% identical to the amino acid sequence of amino acids 1-1228 of SEQ ID NO: 10.

6 . The isolated protein of claim 1 , further comprising mutations at one or more of the following positions: R508, K514, K591, R737, R883, K897, K948, and/or K984.

7 . The isolated protein of claim 6 , wherein the mutations are R508A, K514A, K591A, R737A, R883A, K897A, K948A, and/or K984A.

8 . A fusion protein comprising the isolated protein of claim 1 , fused to a heterologous functional domain, with an optional intervening linker, wherein the Cpf1 protein is capable of interacting with a guide RNA and target DNA.

9 . The fusion protein of claim 8 , wherein the heterologous functional domain is a transcriptional activation domain.

10 . The fusion protein of claim 9 , wherein the transcriptional activation domain is from VP64 or NF-κB p65.

11 . The fusion protein of claim 8 , wherein the heterologous functional domain is a transcriptional silencer or transcriptional repression domain.

12 . The fusion protein of claim 11 , wherein the transcriptional repression domain is a Krueppel-associated box (KRAB) domain, ERF repressor domain (ERD), or mSin3A interaction domain (SID).

13 . The fusion protein of claim 11 , wherein the transcriptional silencer is Heterochromatin Protein 1 (HP1).

14 . The fusion protein of claim 8 , wherein the heterologous functional domain is an enzyme that modifies the methylation state of DNA.

15 . The fusion protein of claim 14 , wherein the enzyme that modifies the methylation state of DNA is a DNA methyltransferase (DNMT) or a TET protein.

16 . The fusion protein of claim 15 , wherein the TET protein is TET1.

17 . The fusion protein of claim 8 , wherein the heterologous functional domain is an enzyme that modifies a histone subunit.

18 . The fusion protein of claim 17 , wherein the enzyme that modifies a histone subunit is a histone acetyltransferase (HAT), histone deacetylase (HDAC), histone methyltransferase (HMT), or histone demethylase.

19 . The fusion protein of claim 8 , wherein the heterologous functional domain is a biological tether.

20 . The fusion protein of claim 19 , wherein the biological tether is MS2, Csy4 or lambda N protein.

21 . The fusion protein of claim 8 , wherein the heterologous functional domain is FokI.

22 . A method of altering the genome of a cell, the method comprising expressing in the cell, or contacting the cell with, the isolated protein or fusion protein of claim 1 , and a guide RNA having a region complementary to a selected portion of the genome of the cell.

23 . The method of claim 22 , wherein the isolated protein or fusion protein comprises one or more of a nuclear localization sequence, cell penetrating peptide sequence, and/or affinity tag.

24 . The method of claim 22 , wherein the cell is a stem cell.

25 . The method of claim 24 , wherein the cell is an embryonic stem cell, mesenchymal stem cell, or induced pluripotent stem cell; is in a living animal; or is in an embryo.

26 . A method of altering a double stranded DNA (dsDNA) molecule, the method comprising contacting the dsDNA molecule with the isolated protein of claim 1 , and a guide RNA having a region complementary to a selected portion of the dsDNA molecule.

27 . The method of claim 26 , wherein the dsDNA molecule is in vitro.

28 . An isolated nucleic acid encoding an isolated Cpf1 protein, wherein the protein is from LbCpf1, comprising a sequence that is at least 80% identical to the amino acid sequence of amino acids 1-1228 of SEQ ID NO: 10, with mutations at one or more of the following positions: N160, S168, N256, N260, K272, S286, K349, D505, Q513, G741, Q944, K945, and/or S985 of amino acids 1-1228 of SEQ ID NO:10.

29 . A vector comprising the isolated nucleic acid of claim 28 .

30 . An isolated host cell, comprising the isolated nucleic acid of claim 28 .

31 . The isolated host cell of claim 30 , wherein the host cell is a mammalian host cell.

32 . The isolated nucleic acid of claim 28 , wherein the sequence is at least 95% identical to the amino acid sequence of amino acids 1-1228 of SEQ ID NO: 10.

33 . The isolated nucleic acid of claim 28 , further comprising mutations at one or more of the following positions: R508, K514, K591, R737, R883, K897, K948, and/or K984.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jan 11, 2022
From: JOUNG, J. KEITH; KLEINSTIVER, BENJAMIN
To: THE GENERAL HOSPITAL CORPORATION
Reel/Frame 058621/0719 →
Continuity (3)
Division 15659499 · Jul 25, 2017
Provisional Application 62366976 · Jul 26, 2016
Related Publication 20220025347A1 · Jan 27, 2022
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