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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.

CRISPR RNA targeting schematic


Mammalian

ID Plasmid Gene/Insert PI Publication
103849pC0039-CMV-dPspCas13b-GS-ADAR2DD(E488Q)PspCas13b (Other), huADAR2DD (Homo sapiens) Zhang RNA editing with CRISPR-Cas13. Science. 2017 Oct 25. pii: eaaq0180. doi: 10.1126/science.aaq0180.
103854pC0043-PspCas13b crRNA backboneU6-PspCas13b DR-BbsI-BbsI-polyT Zhang RNA editing with CRISPR-Cas13. Science. 2017 Oct 25. pii: eaaq0180. doi: 10.1126/science.aaq0180.
103863pC0047-CMV-dPspCas13b-ADAR1DD(E1008Q)PspCas13b (Other), huADAR1DD (Homo sapiens) Zhang RNA editing with CRISPR-Cas13. Science. 2017 Oct 25. pii: eaaq0180. doi: 10.1126/science.aaq0180.
103864pC0048-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q)PspCas13b (Other), huADAR2DD (Homo sapiens) Zhang RNA editing with CRISPR-Cas13. Science. 2017 Oct 25. pii: eaaq0180. doi: 10.1126/science.aaq0180.
103866pC0050-CMV-dPspCas13b-longlinker-ADAR2DD(wt)PspCas13b (Other), huADAR2DD (Homo sapiens) Zhang RNA editing with CRISPR-Cas13. Science. 2017 Oct 25. pii: eaaq0180. doi: 10.1126/science.aaq0180.
103869pC0053-CMV-dPspCas13b-GS-ADAR2DD(E488Q)-delta-984-1090PspCas13b (Other), huADAR2DD (Homo sapiens) Zhang RNA editing with CRISPR-Cas13. Science. 2017 Oct 25. pii: eaaq0180. doi: 10.1126/science.aaq0180.
103870pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)PspCas13b (Other), huADAR2DD (Homo sapiens) Zhang RNA editing with CRISPR-Cas13. Science. 2017 Oct 25. pii: eaaq0180. doi: 10.1126/science.aaq0180.
103871pC0055-CMV-dPspCas13b-GS-ADAR2DD(E488Q/T375G)-delta-984-1090PspCas13b (Other), huADAR2DD (Homo sapiens) Zhang RNA editing with CRISPR-Cas13. Science. 2017 Oct 25. pii: eaaq0180. doi: 10.1126/science.aaq0180.


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