Directed editing of cellular RNA via nuclear delivery of CRISPR/CAS9
AssigneeThe Regents of the University of California
InventorsEugene Yeo, Kristopher Brannan, Ryan Marina, David Nelles
Life of this patent
Estimating the term…
Abstract
Disclosed herein is a technology to perform programmable RNA editing at single-nucleotide resolution using RNA-targeting CRISPR/Cas9. This approach, which Applicants have termed “Cas9-directed RNA editing” or “CREDIT,” provides a means to reversibly alter genetic information in a temporal manner, unlike traditional CRISPR/Cas9 driven genomic engineering which relies on permanently altering DNA sequence.
Claims20
A recombinant expression system for CRISPR/Cas-directed RNA editing of a target RNA comprising:
- (A) a nucleic acid sequence encoding a CRISPR/Cas RNA editing fusion protein comprising a nuclease-dead CRISPR associated endonuclease (dCas) fused to a catalytically active deaminase domain of Adenosine Deaminase acting on RNA 2 (ADAR2);
- and
- (B) a nucleic acid sequence encoding an extended single guide RNA (esgRNA) comprising:
- (i) a short extension sequence of homology to the target RNA comprising a mismatch for a target adenosine, (ii) a dCas scaffold binding sequence, and (iii) a spacer sequence comprising a region of homology to the target RNA, wherein the sequences of the esgRNA (i), (ii), and (iii) are situated 3′ to 5′ in the esgRNA.
A vector comprising a nucleic acid encoding an extended single guide RNA (esgRNA) comprising (i) a short extension sequence of homology to a target RNA comprising a mismatch for a target adenosine, (ii) a dCas scaffold binding sequence, and (iii) a sequence complementary to the target sequence (spacer sequence), wherein (i), (ii) and (iii) are situated 3′ to 5′ in the esgRNA.
A kit comprising:
- (A) one or more selected from the group consisting of:
- (i) a recombinant expression system comprising:
- (a) a nucleic acid sequence encoding a CRISPR/Cas RNA editing fusion protein comprising a nuclease-dead CRISPR associated endonuclease (dCas) fused to a catalytically active deaminase domain of Adenosine Deaminase acting on RNA 2 (ADAR2);
- and
- (b) a nucleic acid sequence encoding an extended single guide RNA (esgRNA) comprising:
- (i) a short extension sequence of homology to the target RNA comprising a mismatch for a target adenosine,
- (ii) a dCas scaffold binding sequence, and
- (iii) a spacer sequence comprising a region of homology to the target RNA,
- wherein the sequences of the esgRNA (i), (ii), and (iii) are situated 3′ to 5′ in the esgRNA;
- (ii) a vector comprising a nucleic acid encoding (A)(i)(a) and (b);
- (iii) a vector comprising a nucleic acid encoding (A)(i)(b);
- (iv) a viral particle comprising the recombinant expression system of (A)(i)(a) and (b);
- (v) an isolated cell comprising the recombinant expression system of (A)(i)(a) and (b);
- and
- (vi) an esgRNA comprising (i) a short extension sequence of homology to a target RNA comprising a mismatch for a target adenosine, and (ii) a dCas scaffold binding sequence, and (iii) a spacer sequence comprising a region of homology to the target RNA, wherein the sequences of the esgRNA (i), (ii), and (iii) are situated 3′ to 5′ in the esgRNA and
- (B) instructions for use.
Dependencies are inferred from the claim text.