Simultaneous multifunctional transcriptome engineering by CRISPR RNA scaffold

Abstract

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. We show that CREST is capable of simultaneously manipulating RNA alternative splicing and A-to-G or C-to-U base editing. Furthermore, by fusing two split fragments of the deaminase domain of ADAR2 to dCas13 and PUFc respectively, we reconstituted its enzyme activity at target sites. This split design can reduce more than 90% 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 and the development of RNA-focused therapeutics.

Customizing cellular signal processing by synthetic multi-level regulatory circuits

“…Altering configurations of UAG codons and downstream CDSs conferred these editing-based riboregulators with the capability to execute RNA-responsive AND, OR logic, memory 76 , and positive feedback 77 . The RNA-editing functions have also been realized by fusing ADAR to dCas13 protein in RCas system 78 . The RCas system could deliver other effector proteins to target RNAs for degradation, translation modulation 79 and alternative splicing 78 .…”

“…The RNA-editing functions have also been realized by fusing ADAR to dCas13 protein in RCas system 78 . The RCas system could deliver other effector proteins to target RNAs for degradation, translation modulation 79 and alternative splicing 78 . Inspired by the RCas system, Rauch et al 80 proposed CIRTS, a minimal RNA targeting system consisting of gRNAs and human protein parts with similar functions but smaller circuit sizes.…”