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

Patent metadata

Field	Value
Publication number	US-11634731-B2
Application number	US-201916572248-A
Country	US
Kind code	B2
Filing date	Sep 16, 2019
Priority date	Mar 15, 2013
Publication date	Apr 25, 2023
Grant date	Apr 25, 2023

Abstract

Methods for increasing specificity of RNA-guided genome editing, e.g., editing using CRISPR/Cas9 systems, using truncated guide RNAs (tru-gRNAs).

First claim

What is claimed is:

1. 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) a S. pyogenes CRISPR dCas9-heterologous functional domain fusion protein (dCas9-HFD) and
(b) 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 target sequence is immediately 5′ of a protospacer adjacent motif (PAM); wherein the guide RNA is:
(i) a single guide RNA that includes a complementarity region at the 5′ end of the single 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 on a double stranded DNA molecule, or
(ii) a crRNA that includes at the 5′ end of the crRNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, and a tracrRNA;

wherein the guide RNA complementarity region binds and directs the dCas9-HFD to the target region of the double-stranded DNA molecule, thereby modifying the target region of a double-stranded DNA molecule in a cell; and
wherein the dCas9-HFD comprises a heterologous functional domain (HFD) that modifies gene expression, histones, or DNA.

2. The method of claim 1 , 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.

3. The method of claim 2 , wherein the HFD is a transcriptional activation domain.

4. The method of claim 3 , wherein the transcriptional activation domain is from activator domain VP64.

5. The method of claim 3 , wherein the transcriptional activation domain is from NF-kappa B subunit p65 (NF-κB p65).

6. The method of claim 2 , wherein the HFD is an enzyme that catalyzes histone modification.

7. The method of claim 6 , wherein the enzyme that catalyzes histone modification is lysine-specific histone demethylase 1 (LSD1).

8. The method of claim 6 , wherein the enzyme that catalyzes histone modification is a histone methyltransferase (HNMT).

9. The method of claim 6 , wherein the enzyme that catalyzes histone modification is histone acetyltransferase (HAT).

10. The method of claim 6 , wherein the enzyme that catalyzes histone modification is histone deacetylase (HDAC).

11. The method of claim 6 , wherein the enzyme that catalyzes histone modification is histone demethylase.

12. The method of claim 2 , wherein the HFD is a transcription silencing domain.

13. The method of claim 12 , wherein the transcription silencing domain is Heterochromatin Protein 1 alpha (HP1α).

14. The method of claim 12 , wherein the transcription silencing domain is Heterochromatin Protein 1 beta (HP1β).

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

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

17. The method of claim 16 , wherein the cell is a mammalian cell.

18. The method of claim 1 , wherein the guide RNA is a single guide RNA that includes a complementarity region at the 5′ end of the single 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 on a double stranded DNA molecule.

19. The method of claim 1 , wherein the guide RNA is a crRNA that includes at the 5′ end of the crRNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, and a tracrRNA.

Assignees

• Massachusetts Gen Hospital

Inventors

• Joung J Keith
• Sander Jeffry D
• Maeder Morgan
• Fu Yanfang

Classifications

• C07K2319/00

  Fusion polypeptide · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N15/01

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

• C12Y114/11

  with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11) · CPC title

• C12N9/22

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