Increasing Specificity for RNA-Guided Genome Editing

• US 20180208921A1
• Filed: 01/12/2018
• Published: 07/26/2018
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Joung, J. Keith, Angstman, James, Sander, Jeffry D., Maeder, Morgan, Tsai, Shengdar
• Granted Patent: US 10,844,403 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/01 containing a localisation/targetting motif
• 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 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/11 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 Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2710/00033 Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
• CPC Class Codes: C12N 2770/00033 Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/1007 Methyltransferases (general) (2.1.1.)
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/96 : Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Y 114/11 : with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
• CPC Class Codes: C12Y 201/01 : Methyltransferases (2.1.1)
• CPC Class Codes: C12Y 301/00 : Hydrolases acting on ester bonds (3.1)
• CPC Class Codes: C12Y 301/21004 : Type II site-specific deoxyribonuclease (3.1.21.4)

Abstract

Methods for increasing specificity of RNA-guided genome editing, e.g., editing using CRISPR/Cas9 systems.

Claims

1. A hybrid guide nucleic acid consisting of the sequence:

2. A vector comprising the DNA molecule of claim 1.

3. A host cell expressing the hybrid guide nucleic acid of claim 1.

4. The hybrid guide nucleic acid of claim 1, wherein the one or more deoxyribonucleotides are within the sequence complementary to 17-20 consecutive nucleotides of the complementary strand of the target sequence.

5. The hybrid guide nucleic acid of claim 1, wherein the one or more deoxyribonucleotides comprise thymine in place of uracil.

6. The hybrid guide nucleic acid of claim 1, wherein the X_17-20 is at least partially or wholly DNA.

27. The hybrid guide nucleic acid of claim 1, wherein N is 0-20.

28. The hybrid guide nucleic acid of claim 1, wherein N is 0.

7. A composition comprising:

a nucleic acid encoding a variant S. pyogenes Cas9 protein comprising an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO;

18 with mutations at D10, E762, H983, D986, H840 or N863; and

a nucleic acid encoding a hybrid guide nucleic acid that directs the variant S. pyogenes Cas9 protein to a target sequence;

wherein the hybrid guide nucleic acid is selected from the group consisting of;

8. The composition of claim 7, wherein the one or more deoxyribonucleotides are within the sequence complementary to 17-20 consecutive nucleotides of the complementary strand of the target sequence.

9. The composition of claim 7, wherein the one or more deoxyribonucleotides comprise thymine in place of uracil.

10. The composition of claim 7, wherein the X_17-20 is at least partially or wholly DNA.

11. The composition of claim 7, wherein the variant S. pyogenes Cas9 comprises a mutation at positions D10 and H840.

12. The composition of claim 11, wherein the mutation at position D10 is D10A or D1 ON, and the mutation at position H840 is H840A, H840N or H840Y.

13. The composition of claim 7, wherein the variant S. pyogenes Cas 9 protein is fused to a heterologous functional domain, with an optional intervening linker.

14. The composition of claim 13, wherein the heterologous functional domain is FokI.

15. The composition of claim 13, wherein the heterologous functional domain is a transcriptional activation domain.

16. The composition of claim 15, wherein the transcriptional activation domain is from VP64 or NK-κ

B p65.

17. The composition of claim 13, wherein the heterologous functional domain is a transcriptional silencer or a transcriptional repression domain.

18. The composition of claim 17, wherein the transcriptional repression domain is a Krueppel-associated box (KRAB) domain, ERF repressor domain (ERD), or mSin3A interaction domain (SID).

19. The composition of claim 18, wherein the transcriptional silencer is Heterochromatin Protein 1 (HP1).

20. The composition of claim 13, wherein the heterologous functional domain is an enzyme that modifies the methylation state of DNA.

21. The composition of claim 20, wherein the enzyme that modifies the methylation state of DNA is a DNA methyltransferase (DNMT) or a Ten-Eleven-Translocation (TET) protein.

22. The composition of claim 21, wherein the TET protein is TET1.

23. The composition of claim 13, wherein the heterologous functional domain is an enzyme that modifies a histone subunit.

24. The composition of claim 23, wherein the enzyme that modifies a histone subunit is a histone acetyltransferase (HAT), a histone deacetylase (HDAC), a histone methyltransferase (HMT), or a histone demethylase.

25. The composition of claim 13, wherein the heterologous functional domain is a biological tether.

26. The composition of claim 25, wherein the biological tether is MS2, CRISPR/Cas Subtype Ypest protein 4 (Csy4), or lambda N protein.

29. The composition of claim 7, wherein N is 0-20.

30. The composition of claim 7, wherein N is 0.