Methods for nucleic acid editing

• US 9,840,699 B2
• Filed: 07/08/2014
• Issued: 12/12/2017
• Est. Priority Date: 12/12/2013
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Liu, David R., Komor, Alexis Christine
• Primary Examiner(s): KIM, ALEXANDER D
• Application Number:
  US14/326,109
• Publication Number:
  US 20150166981A1
• Time in Patent Office: 1,253 Days
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• CPC Class Codes: A61K 38/50 acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
• CPC Class Codes: A61K 47/61 the organic macromolecular compound being a polysaccharide or a derivative thereof
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/02 of urine or of the urinary tract, e.g. urine acidifiers
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 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 Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/6472 Cysteine endopeptidases (3.4.22)
• CPC Class Codes: C12N 9/78 acting on carbon to nitrogen bonds other than peptide bonds (3.5)
• CPC Class Codes: C12P 19/34 : Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6883 : for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/156 : Polymorphic or mutational markers
• CPC Class Codes: C12Y 301/00 : Hydrolases acting on ester bonds (3.1)
• CPC Class Codes: C12Y 301/22 : Endodeoxyribonucleases producing 3'-phosphomonoesters (3.1.22)
• CPC Class Codes: C12Y 304/22062 : Caspase-9 (3.4.22.62)
• CPC Class Codes: C12Y 305/04 : in cyclic amidines (3.5.4)
• CPC Class Codes: C12Y 305/04001 : Cytosine deaminase (3.5.4.1)
• CPC Class Codes: C12Y 305/04004 : Adenosine deaminase (3.5.4.4)
• CPC Class Codes: C12Y 305/04005 : Cytidine deaminase (3.5.4.5)
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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.

Claims

1. A method of DNA editing, the method comprising contacting a DNA molecule with

(a) a fusion protein comprising (i) a nuclease-inactive Cas9 (dCas9) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

4, wherein the dCas9 comprises an alanine at a position corresponding to position 10 in SEQ ID NO;

4, and (ii) a cytidine deaminase; and

(b) a single-guide RNA (sgRNA) targeting the fusion protein of (a) to a target nucleotide sequence of the DNA molecule, comprising the nucleotide sequence of 5′

-[a guide sequence]-guuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu-3′

(SEQ ID NO;

     38), wherein the guide sequence comprises a RNA sequence that is complementary to the target nucleotide 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 a deamination of a nucleotide base, wherein the method results in the deamination of a nucleotide base within the DNA molecule.

2. The method of claim 1, wherein the cytidine deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase.

3. The method of claim 2, wherein the cytidine deaminase is an APOBEC1 family deaminase.

4. The method of claim 3, wherein the cytidine deaminase is an activation-induced cytidine deaminase (AID).

5. The method of claim 3, wherein the deaminase is an APOBEC1 deaminase comprising the amino acid sequence of SEQ ID NO:

22, SEQ ID NO;

23, or SEQ ID NO;

24.

6. The method of claim 1, wherein the cytidine deaminase is fused to the N-terminus of the dCas9.

7. The method of claim 1, wherein the cytidine deaminase is fused to the C-terminus of the dCas9.

8. The method of claim 1, wherein the dCas9 and the cytidine deaminase are fused via a linker.

9. The method of claim 8, wherein the linker comprises a (GGGGS)_n (SEQ ID NO:

91), a (G)_n, an (EAAAK)_n (SEQ ID NO;

     5), or an (XP)_n motif, or a combination of any of these, wherein n is independently an integer between 1 and 30.

10. The method of claim 1, 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.

11. The method of claim 10, wherein the deamination corrects a point mutation in the sequence associated with the disease or disorder.

12. The method of claim 10, 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.

13. The method of claim 10, wherein the contacting is in vivo in a subject having or diagnosed with the disease or disorder.

14. The method of claim 10, wherein the disease 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), or a neoplastic disease associated with a mutant PI3KCA protein.

15. The method of claim 1, wherein the DNA sequence comprises a T to C point mutation associated with a disease or disorder, and wherein the deamination of the mutant C base results in a sequence that is not associated with a disease or disorder.

16. The method of claim 1, wherein the dCas9 of (a) further comprises one or more mutations corresponding to a D839A or a N863A in SEQ ID NO:

4.

17. The method of claim 1, wherein the DNA sequence encodes a protein, and wherein deamination of the nucleotide base results in a correction of a T to C point mutation to restore the wild-type sequence of the encoded protein.

18. The method of claim 1, wherein the guide sequence is 20 nucleotides long.

19. The method of claim 1, wherein the dCas9 comprises the amino acid sequence of SEQ ID NO:

4.

20. The method of claim 1, wherein the nucleotide base of the DNA molecule deaminated by the fusion protein is located on a nucleotide strand complementary to the target nucleotide sequence of the DNA molecule that is to contacted by the guide sequence of the sgRNA.