Increasing specificity for RNA-guided genome editing

Patent metadata

Field	Value
Publication number	US-10844403-B2
Application number	US-201815870659-A
Country	US
Kind code	B2
Filing date	Jan 12, 2018
Priority date	Mar 15, 2013
Publication date	Nov 24, 2020
Grant date	Nov 24, 2020

Abstract

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

First claim

What is claimed is:

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

(SEQ ID NO: 4)

(X 17-20 )GUUUUAGAGCUAUGCUGUUUUG(X N );

(SEQ ID NO: 5)

(X 17-20 )GUUUUAGAGCUA;

(SEQ ID NO: 6)

(X 17-20 )GUUUUAGAGCUAUGCUGUUUUG;

(SEQ ID NO: 7)

(X 17-20 )GUUUUAGAGCUAUGCU;

(SEQ ID NO: 8)

(X 17-20 )GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG

UCCG(X N );

(SEQ ID NO: 9)

(X 17-20 )GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAU

AAGGCUAGUCCGUUAUC(X N );

(SEQ ID NO: 10)

(X 17-20 )GUUUUAGAGCUAUGCUGUUUUGGAAACAAAACAGCAUA

GCAAGUUAAAAUAAGGCUAGUCCGUUAUC(X N );

(SEQ ID NO: 11)

(X 17-20 )GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG

UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X N );

(SEQ ID NO: 12)

(X 17-20 )GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAG

UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC;

(SEQ ID NO: 13)

(X 17-20 )GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAA

AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG

GUGC;

and

(SEQ ID NO: 14)

(X 17-20 )GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAA

AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG

GUGC,

wherein the X 17-20 is a sequence complementary to 17-20 consecutive nucleotides of the complementary strand of a target sequence, preferably a target sequence immediately 5′ of a protospacer adjacent motif (PAM), wherein one or more of the nucleotides is a deoxyribonucleic acid, and N is 0-50.

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.

7. A composition comprising:
a nucleic acid encoding a variant S. pyogenes Cas 9 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:
(X 17 -20) GUUUUAGAGCUAUGCUGUUUUG(XN) (SEQ ID NO:4);
(X 17 -20) GUUUUAGAGCUA (SEQ ID NO:5);
(X 17 -20) GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO:6);
(X 17 -20) GUUUUAGAGCUAUGCU (SEQ ID NO:7);
(X 17 -20) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG(XN) (SEQ ID NO:8);
(X 17 -20) GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUC(XN) (SEQ ID NO:9);
(X 17 -20) GUUUUAGAGCUAUGCUGUUUUGGAAACAAAACAGCAUAGCAAGUU AAAAUAAGGCUAGUCCGUUAUC(XN) (SEQ ID NO:10);
(X 17 -2o)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUA UCAACUUGAAAAAGUGGCACCGAGUCGGUGC(XN) (SEQ ID NO:11);
(X 17 -2o)GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(SEQ ID NO:12);
(X 17 -2o)GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:13); and
(X 17 -2o)GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGC UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:14),
wherein the X 17 -20 is a sequence complementary to 17-20 consecutive nucleotides of the complementary strand of a target sequence, preferably a target sequence immediately 5′ of a protospacer adjacent motif (PAM), wherein one or more of the nucleotides is a deoxyribonucleic acid, and N is 0-50.

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 Cas 9 comprises a mutation at positions D10 and H840.

12. The composition of claim 11 , wherein the mutation at position D10 is D10A or D10N, 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

Assignees

• Massachusetts Gen Hospital

Inventors

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

Classifications

• C12Y301/00

  Hydrolases acting on ester bonds (3.1) · CPC title

• C12Y201/01

  Methyltransferases (2.1.1) · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · 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

• C12N15/102

  Mutagenizing nucleic acids · CPC title