Increasing specificity for RNA-guided genome editing

• US 9,885,033 B2
• Filed: 03/14/2014
• Issued: 02/06/2018
• 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
• Primary Examiner(s): Bowman, Amy
• Application Number:
  US14/776,620
• Publication Number:
  US 20160024524A1
• Time in Patent Office: 1,425 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 method of inducing a single or double-stranded break in a target region of a double-stranded DNA molecule in a cell, the method comprising expressing in or introducing into the cell:

a Streptococcus pyogenes Cas9 nuclease or nickase; and

a guide RNA that consists of a sequence selected from the group consisting of;

2. The method of claim 1, wherein the one or more deoxyribonucleotides comprise thymine in place of uracil.

3. The method 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.