Simultaneous multifunctional transcriptome engineering by CRISPR RNA scaffold

RNA processing and metabolism are subjected to precise regulation in the cell to ensure integrity and functions of RNA. Though targeted RNA engineering has become feasible with the discovery and engineering of the CRISPR-Cas13 system, simultaneous modulation of different RNA processing steps remains unavailable. In addition, off-target events resulting from effectors fused with dCas13 limit its application. Here we developed a novel platform, Combinatorial RNA Engineering via Scaffold Tagged gRNA (CREST), which can simultaneously execute multiple RNA modulation functions on different RNA targets. In CREST, RNA scaffolds are appended to the 3' end of Cas13 gRNA and their cognate RNA binding proteins are fused with enzymatic domains for manipulation. Taking RNA alternative splicing, A-to-G and C-to-U base editing as examples, we developed bifunctional and tri-functional CREST systems for simultaneously RNA manipulation. Furthermore, by fusing two split fragments of the deaminase domain of ADAR2 to dCas13 and/or PUFc respectively, we reconstituted its enzyme activity at target sites. This split design can reduce nearly 99% of off-target events otherwise induced by a full-length effector. The flexibility of the CREST framework will enrich the transcriptome engineering toolbox for the study of RNA biology.

Automated summary

We developed a novel platform called CREST that can simultaneously execute multiple RNA modulation functions on different RNA targets. By appending RNA scaffolds to Cas13 gRNA and fusing cognate RNA binding proteins with enzymatic domains, we achieved manipulation of RNA. Additionally, we reconstituted the enzyme activity of ADAR2 at target sites by fusing split fragments of its deaminase domain to dCas13 and/or PUFc, reducing off-target events significantly.