Increasing Specificity for RNA-Guided Genome Editing

Patent metadata

Field	Value
Publication number	US-2021130850-A1
Application number	US-202017099503-A
Country	US
Kind code	A1
Filing date	Nov 16, 2020
Priority date	Mar 15, 2013
Publication date	May 6, 2021
Grant date	—

Abstract

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

First claim

1 .- 30 . (canceled)

31 . A method of inducing a single or double-stranded break in a target region of a double-stranded DNA molecule the method comprising expressing in or introducing into the cell:
a Cas9 nuclease or nickase; a tracrRNA; and a crRNA that includes one or more nucleotides that are modified, wherein one or more of the nucleotides in the target complementarity region is modified, and wherein the crRNA includes a sequence of 17-20 nucleotides that are complementary to the target sequence, preferably the target sequence immediately 5′ of a protospacer adjacent motif (PAM).

32 . The method of claim 31 , wherein the one or more nucleotides that are modified are selected from 2′-O-4′-C methylene bridge, 5′-methylcytidine, or 2′-O-methyl-pseudouridine.

33 . The method of claim 31 , wherein the one or more nucleotides that are modified and wherein the ribose phosphate backbone has been replaced by a polyamide chain.

34 . The method of claim 31 , wherein the crRNA consists of the sequence: (X17 20)GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO:4);
(X17 20)GUUUUAGAGCUA (SEQ ID NO:5); (X17 20)GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO:6); or (X17 20)GUUUUAGAGCUAUGCU (SEQ ID NO:7).

35 . The method of claim 34 , wherein X17 20 is not identical to a sequence that naturally occurs adjacent to the rest of the RNA.

36 . The method of claim 34 , wherein the crRNA includes one or more U at the 3′ end of the molecule.

37 . The method of claim 34 , wherein the crRNA includes one or more additional nucleotides at the 5′ end of the RNA molecule that is not complementary to the target sequence.

38 . The method of claim 31 , wherein the tracrRNA comprises the sequence of:
GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC UUGAAAAAGUGGCACCGAGTCGGUGCUUUU (SEQ ID NO:15) or an active portion thereof, UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC GAGUCGGUGC (SEQ ID NO:16) or an active portion thereof, AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGG CACCGAGUCGGUGC (SEQ ID NO:17) or an active portion thereof, GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC AACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:42) or an active portion thereof, UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC GAGUCGGUGC (SEQ ID NO:16) or an active portion thereof, CAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA AAGUGGCACCGAGUCGGUGC (SEQ ID NO:43) or an active portion thereof; AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGG CACCGAGUCGGUGC (SEQ ID NO:17) or an active portion thereof; UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG (SEQ ID NO:44) or an active portion thereof; UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA (SEQ ID NO:45) or an active portion thereof; or UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO:46) or an active portion thereof.

39 . The method of claim 31 , wherein the crRNA has the sequence of:

(SEQ ID NO: 6)

(X17-20)GUUUUAGAGCUAUGCUGUUUUG,

and the tracrRNA has the sequence of: GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU UGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:8) or an active portion thereof.

40 . The method of claim 31 , wherein the crRNA has the sequence of:
(X17-20)GUUUUAGAGCUA (SEQ ID NO:5), and the tracrRNA has the sequence of: UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC GAGUCGGUGC (SEQ ID NO:16) or an active portion thereof.

41 . The method of claim 31 , wherein the crRNA has the sequence of:
(X17-20) GUUUUAGAGCUAUGCU (SEQ ID NO:7), and the tracrRNA has the sequence of: AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC GAGUCGGUGC (SEQ ID NO:17) or an active portion thereof.

Assignees

• Massachusetts Gen Hospital

Inventors

• Joung J Keith
• Angstman James
• Sander Jeffry D
• Maeder Morgan
• Tsai Shengdar

Classifications

• C12Y201/01

  Methyltransferases (2.1.1) · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12Y301/00

  Hydrolases acting on ester bonds (3.1) · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/113 Primary

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title