Using truncated guide RNAs (tru-gRNAs) to increase specificity for RNA-guided genome editing

Patent metadata

Field	Value
Publication number	US-10119133-B2
Application number	US-201414775930-A
Country	US
Kind code	B2
Filing date	Mar 14, 2014
Priority date	Mar 15, 2013
Publication date	Nov 6, 2018
Grant date	Nov 6, 2018

Abstract

CRISPR-Cas genome editing uses a guide RNA, which includes both a complementarity region, which binds the target DNA by base-pairing, and a Cas9-binding region, to direct a Cas9 nuclease to a target DNA. Further disclosed are methods for increasing specificity of RNA-guided genome editing using CRISPR/Cas9 systems by using truncated guide RNAs (tru-gRNAs).

First claim

What is claimed is:

1. A method of increasing specificity of S. pyogenes CRISPR-Cas9 (Cas9) RNA-guided genome editing in a cell, the method comprising contacting the cell with a guide RNA that includes a complementarity region at the 5′ end of the guide RNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, wherein the selected target genomic sequence is immediately 5′ of a protospacer adjacent motif (PAM), the guide RNA comprises
SEQ ID NO:4, and wherein in the presence of a S. pyogenes Cas9 genome editing enzyme, the guide RNA complementarity region binds and directs the Cas9 genome editing enzyme to the selected target genomic sequence, thereby increasing specificity of RNA-guided genome editing in a cell.

2. A method of inducing a 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 S. pyogenes CRISPR/Cas9 nuclease or nickase; and
a guide RNA that includes a complementarity region at the 5′end of the guide RNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a double-stranded DNA molecule comprising a target sequence, wherein the target sequence is immediately 5′ of a protospacer adjacent motif (PAM), and wherein the guide RNA complementarity region binds and directs the Cas9 nuclease or nickase to the target region of a double-stranded DNA molecule, and wherein the guide RNA comprises
SEQ ID NO:4, thereby inducing a break in the target region of a double-stranded DNA molecule in a cell.

3. A method of modifying a target region of a double-stranded DNA molecule in a cell, the method comprising expressing in or introducing into the cell:
a S. pyogenes CRISPR dCas9-heterologous functional domain fusion protein (dCas9-HFD); and
a guide RNA that includes a complementarity region at the 5′end of the guide RNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target sequence present on a double-stranded DNA molecule, wherein the selected target sequence is immediately 5′ of a protospacer adjacent motif (PAM), the guide RNA comprises
SEQ ID NO:4, and wherein the guide RNA complementarity region binds and directs the dCas9-HFD to the selected target sequence, thereby modifying a target region of a double-stranded DNA molecule in a cell.

4. The method of claim 1 , wherein the target region is in a target genomic sequence.

5. The method of claim 3 , wherein dCas9-HFD comprises a heterologous functional domain (HFD) that modifies gene expression, histones, or DNA.

6. The method of claim 5 , wherein the HFD is a transcriptional activation domain, an enzyme that catalyzes DNA demethylation, an enzyme that catalyzes histone modification, or a transcription silencing domain.

7. The method of claim 6 , wherein the transcriptional activation domain is from activator domain VP64 or NF-kappa B subunit p65 (NF-κB p65).

8. The method of claim 6 , wherein the enzyme that catalyzes histone modification is lysine-specific histone demethylase 1 (LSD1), a histone methyltransferase (HNMT), histone acetyltransferase (HAT), histone deacetylase (HDAC), or histone demethylase.

9. The method of claim 6 , wherein the transcription silencing domain is from Heterochromatin Protein 1 alpha (HP1α) or Heterochromatin Protein 1 beta (HP1β).

10. The method of claim 1 , wherein the cell is a eukaryotic cell.

11. The method of claim 10 , wherein the cell is a mammalian cell.

Assignees

• Massachusetts Gen Hospital

Inventors

• Joung J Keith
• Sander Jeffry D
• Fu yan-fang
• Maeder Morgan

Classifications

• C12N9/22

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

• C12N15/1031

  mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR · CPC title

• C12N15/01

  Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor · CPC title

• C12Y201/01

  Methyltransferases (2.1.1) · CPC title

• C12N15/11

  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 title