Using RNA-guided FokI Nucleases (RFNs) to Increase Specificity for RNA-Guided Genome Editing

Patent metadata

Field	Value
Publication number	US-2020224222-A1
Application number	US-202016751578-A
Country	US
Kind code	A1
Filing date	Jan 24, 2020
Priority date	Mar 15, 2013
Publication date	Jul 16, 2020
Grant date	—

Abstract

Many studies have shown that CRISPR-Cas nucleases can tolerate up to five mismatches and still cleave; it is hard to predict the effects of any given single or combination of mismatches on activity. Taken together, these nucleases can show significant off-target effects but it can be challenging to predict these sites. Described herein are methods for increasing the specificity of genome editing using the CRISPR/Cas system, e.g., using RNA-guided FokI Nucleases (RFNs), e.g., FokI-Cas9 or FokI-dCas9-based fusion proteins.

First claim

1 .- 16 . (canceled)

17 . An RNA-guided FokI Nuclease (RFN) fusion protein comprising SEQ ID NO:24.

18 . A nucleic acid encoding the fusion protein of claim 17 .

19 . The nucleic acid of claim 18 , comprising SEQ ID NO:27.

20 . A vector comprising the nucleic acid of claim 18 .

21 . A host cell expressing the fusion protein of claim 17 .

22 . A method of inducing a sequence-specific break in a genomic sequence in a cell, the method comprising expressing in the cell, or contacting the cell with, the RNA-guided FokI Nuclease (KEN) fusion protein of claim 17 , and guide RNAs that direct the RFN to two target genomic sequences.

23 . The method of claim 22 , wherein the guide RNAs are:
(a) two single guide RNAs, wherein one single guide RNA targets a first strand, and the other guide RNA targets the complementary strand, and FokI cuts each strand resulting in a pair of nicks on opposite DNA strands, thereby creating a double-stranded break, or (b) a tracrRNA and two crRNAs wherein one crRNA targets a first strand, and the other crRNA targets the complementary strand, and FokI cuts each strand resulting in a pair of nicks on opposite DNA strands, thereby creating a double-stranded break.

24 . The method of claim 22 , wherein each of the two guide RNAs include a complementarity region that is complementary to 17 - 20 nucleotides of target genomic sequence.

25 . The method of claim 22 , wherein an indel mutation is induced between the two target sequences.

26 . The method of claim 22 , wherein the two target genomic sequences each have a Protospacer Adjacent Motif (PAM) sequence at the 3′ end.

27 . The method of claim 22 , wherein the two target genomic sequences are spaced 0-31 nucleotides apart.

28 . The method of claim 27 , wherein the two target genomic sequences are spaced 10-20 base pairs apart.

29 . The method of claim 28 , wherein the two target genomic sequences are spaced 13-17 base pairs apart.

30 . A method of increasing specificity of RNA-guided genome editing in a cell, the method comprising contacting the cell with the RNA-guided FokI Nuclease (RFN) fusion protein of claim 17 .

Assignees

• Massachusetts Gen Hospital

Inventors

• Joung J Keith
• 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