Cas variants for gene editing

Patent metadata

Field	Value
Publication number	US-2016304846-A1
Application number	US-201415103608-A
Country	US
Kind code	A1
Filing date	Dec 12, 2014
Priority date	Dec 12, 2013
Publication date	Oct 20, 2016
Grant date	—

Abstract

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing enzymes or domains, are provided.

First claim

What is claimed is:

1 . A fusion protein comprising
(i) a nuclease-inactive Cas9 domain; and (ii) a nucleic acid-editing domain.

2 . The fusion protein of claim 1 , wherein the nucleic acid-editing domain is a DNA-editing domain.

3 . The fusion protein of claim 1 , wherein the nucleic acid-editing domain is a deaminase domain

4 . The fusion protein of claim 1 , wherein the deaminase is a cytidine deaminase.

5 . The fusion protein of claim 1 , wherein the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase.

6 . The fusion protein of claim 5 , wherein the deaminase is an APOBEC1 family deaminase.

7 . The fusion protein of claim 5 , wherein the deaminase is an activation-induced cytidine deaminase (AID).

8 . The fusion protein of claim 1 , wherein the deaminase is an ACF1/ASE deaminase.

9 . The fusion protein of claim 1 , wherein the deaminase is an adenosine deaminase.

10 . The fusion protein of claim 9 , wherein the deaminase is an ADAT family deaminase.

11 . The fusion protein of claim 1 , wherein the nucleic acid-editing domain is fused to the N-terminus of the Cas9 domain.

12 . The fusion protein of claim 1 , wherein the nucleic acid-editing domain is fused to the C-terminus of the Cas9 domain.

13 . The fusion protein of claim 1 , wherein the Cas9 domain and the nucleic acid-editing domain are fused via a linker.

14 . The fusion protein of claim 1 , wherein the linker comprises a (GGGGS) n (SEQ ID NO: 91), a (G) n , an (EAAAK) n (SEQ ID NO: 5), a (GGS) n , an SGSETPGTSESATPES (SEQ ID NO: 93), or an (XP) n motif, or a combination of any of these, wherein n is independently an integer between 1 and 30.

15 . A method of DNA editing, the method comprising contacting a DNA molecule with
(a) a fusion protein comprising a nuclease-inactive Cas9 domain and a nucleic acid-editing domain; and (b) an sgRNA targeting the fusion protein of (a) to a target DNA sequence of the DNA molecule; wherein the DNA molecule is contacted with the fusion protein and the sgRNA in an amount effective and under conditions suitable for the deamination of a nucleotide base of the DNA molecule.

16 . The method of claim 15 , wherein the nucleic acid-editing domain is a deaminase domain.

17 . The method of claim 16 , wherein the deaminase is a cytidine deaminase.

18 . The method of any one of claim 16 or 17 , wherein the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase.

19 . The method of claim 16 , wherein the deaminase is an APOBEC1 family deaminase.

20 . The method of claim 16 , wherein the deaminase is an activation-induced cytidine deaminase (AID).

21 . The method of claim 16 , wherein the deaminase is an ACF1/ASE deaminase.

22 . The method of claim 16 , wherein the deaminase is an adenosine deaminase.

23 . The method of claim 16 , wherein the deaminase is an ADAT family deaminase.

24 . The method of any one of claims 15 - 23 , wherein the nucleic acid-editing domain is fused to the N-terminus of the Cas9 domain.

25 . The method of any one of claims 15 - 23 , wherein the nucleic acid-editing domain is fused to the C-terminus of the Cas9 domain.

26 . The method of any one of claims 15 - 25 , wherein the Cas9 domain and the nucleic acid-editing domain are fused via a linker.

27 . The method of claim 26 , wherein the linker comprises a (GGGGS) n (SEQ ID NO: 91), a (G) n , an (EAAAK) n (SEQ ID NO: 5), a (GGS) n , an SGSETPGTSESATPES (SEQ ID NO: 93), or an (XP) n motif, or a combination of any of these, wherein n is independently an integer between 1 and 30.

28 . The method of any one of claims 15 - 27 , wherein the target DNA sequence comprises a sequence associated with a disease or disorder, and wherein the deamination of the nucleotide base results in a sequence that is not associated with a disease or disorder.

29 . The method of claim 28 , wherein the target DNA sequence comprises a point mutation associated with a disease or disorder, and wherein the deamination corrects the point mutation.

30 . The method of any one of claims 15 - 28 , wherein the target DNA sequence comprises a T→C or A→G point mutation associated with a disease or disorder, and wherein the deamination of the mutant C or G base results in a sequence that is not associated with a disease or disorder.

31 . The method of any one of claims 28 - 30 , wherein the sequence associated with the disease or disorder encodes a protein, and wherein the deamination introduces a stop codon into the sequence associated with the disease or disorder, resulting in a truncation of the encoded protein.

32 . The method of any one of claims 28 - 31 , wherein the contacting is in vivo in a subject having or diagnosed with a disease or disorder.

33 . The method of any one of claims 28 - 32 , wherein the disease or disorder is cystic fibrosis, phenylketonuria, epidermolytic hyperkeratosis (EHK), Charcot-Marie-Toot disease type 4J, neuroblastoma (NB), von Willebrand disease (vWD), myotonia congenital, hereditary renal amyloidosis, dilated cardiomyopathy (DCM), hereditary lymphedema, familial Alzheimer's disease, Prion disease, chronic infantile neurologic cutaneous articular syndrome (CINCA), desmin-related myopathy (DRM), or a neoplastic disease associated with a mutant PI3KCA protein.

34 . The method of any one of claims 15 - 33 , wherein the target DNA sequence comprises a T→C and/or an A→G point mutation that results in an amino acid sequence mutation in the PI3KCA protein as compared to the wild type PI3K protein, and wherein the method results in the deamination of the mutant C or G base.

35 . The method of claim 34 , wherein the point mutation is an A3140G mutation resulting in an H1047R substitution.

36 . The method of any one of claims 15 - 33 , wherein the target DNA sequence comprises a T→C and/or an A→G point mutation that results in an amino acid sequence mutation in the PSEN1 protein as compared to the wild type PSEN1 protein, and wherein the method results in the deamination of the mutant C or G base.

37 . The method of claim 36 , wherein the PSEN1 protein comprises an I143V substitution caused by an A→G point mutation in codon 143 of the PSEN1 gene.

38 . The method of claim 36 or 37 , wherein the PSEN1 point mutation is associated with Alzheimer's disease.

39 . The method of any one of claims 36 - 38 , wherein the contacting results in deamination of the mutant cytidine residue in codon 143 of the PSEN1 gene, thus correcting the A→G point mutation.

40 . The method of any one of claims 15 - 33 , wherein the target DNA sequence comprises a T→C and/or an A→G point mutation that results in an amino acid sequence mutation in the α-antitrypsin protein as compared to the wild type α-antitrypsin protein, and wherein the method results in the deamination of the mutant C or G base.

41 . The method of claim 40 , wherein the α-antitrypsin protein comprises an L55P substitution caused by an T→C point mutation in codon 55 of the α-antitrypsin gene.

42 . The method of claim 40 or 41 , wherein the α-antitrypsi

Assignees

• Harvard College

Inventors

• Liu David R
• Komor Alexis Christine

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• A61P3/00

  Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00) · CPC title

• A61P25/28

  for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia · CPC title

• A61P17/00

  Drugs for dermatological disorders · CPC title

• A61P11/00

  Drugs for disorders of the respiratory system · CPC title