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.

RNA加工和代谢在细胞中受到精确调控，以确保RNA的完整性和功能。尽管随着CRISPR - Cas13系统的发现和改造，靶向RNA工程已成为可能，但对不同RNA加工步骤的同时调控仍然无法实现。此外，与dCas13融合的效应物导致的脱靶事件限制了其应用。在此，我们开发了一个新的平台，即通过支架标记的gRNA进行组合式RNA工程（CREST），它能够对不同的RNA靶点同时执行多种RNA调控功能。在CREST中，RNA支架被添加到Cas13 gRNA的3’末端，并且它们的同源RNA结合蛋白与酶结构域融合以进行操作。以RNA可变剪接、A到G和C到U碱基编辑为例，我们开发了双功能和三功能的CREST系统用于同时进行RNA操作。此外，通过将ADAR2的脱氨酶结构域的两个分裂片段分别与dCas13和/或PUFc融合，我们在靶点处重建了其酶活性。这种分裂式设计可以减少近99%由全长效应物诱导的脱靶事件。CREST框架的灵活性将丰富用于RNA生物学研究的转录组工程工具箱。