Abstract
1. 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) 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.
| Original language | English (US) |
|---|---|
| Patent number | 11453891 |
| IPC | C12N9/78 |
| Priority date | 5/10/17 |
| Filing date | 5/9/18 |
| State | Published - Sep 27 2022 |