Simultaneous multifunctional transcriptome engineering by CRISPR RNA scaffold. Nucleic Acids Res. 2023;51(14):e77.
JGM, JMG, RNA, CRISPR-Cas Systems, Transcriptome, RNA Processing, Post-Transcriptional, RNA Splicing, Gene Editing
Nucleic Acids Res. 2023;51(14):e77.
National Human Genome Research Institute [R01- HG009900 to A.W.C.]; laboratory startup funds (to P.R.). Funding for open access charge: NIH/NHGRI [R01-HG009900].
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.