CRISPR Plasmids: RNA Editing
Type VI CRISPR systems, including the enzymes Cas13a/C2c2 and Cas13b, target RNA rather than DNA. Fusing the catalytic domain of ADAR2(E488Q) adenosine deaminase to catalytically dead Cas13b creates a programmable RNA editor that converts adenosine to inosine in RNA. Since inosine is functionally equivalent to guanosine, the result is an A->G change in RNA. dPspCas13b does not require a Protospacer Flanking Sequence (PFS), making it a very flexible editing system. The T375G mutation reduces off-target effects of ADAR2 by destabilizing its RNA binding. Editors carrying the delta-984-1090 ADAR2 truncation retain RNA editing capabilities but are small enough to be packaged in AAV particles.