Abstract
Cas13 endonuclease activity depends on the RNA local secondary structure with strong preference for single-stranded (SS) regions. Hence, it becomes indispensable to identify the SS regions for effective Cas13 mediated RNA knockdown. We herein present rational gRNA design by integrating experimental structure-seq data and predicted structural models. Utilizing structure-seq data for XIST transcript, we observed that gRNAs targeting the SS regions significantly induce transcript knockdown and cleavage than those targeting double-stranded (DS) regions. Further, we identified the “central seed region” in the gRNA that upon targeting the SS regions efficiently facilitates Cas13 mediated cleavage. In our following pursuits, we considered the scenario wherein experimental structure-seq data is not available, hence we used SS18-SSX2 fusion transcript indicated in synovial sarcomas and computationally predicted its structure. We observed that gRNAs targeting the SS regions predicted from the structure, efficiently induced necrosis compared to gRNAs that target the DS regions. In conclusion, for the effective RNA knockdown, the Cas13 mediated targeting strategy presented herein emphasizes the designing of gRNAs specifically targeting SS regions by utilizing structural information. Further, this strategy, in turn, can be anticipated to narrow the search space for gRNA design (by exclusively targeting SS regions) especially when lncRNAs are the targets.
Highlights
Bacterial CRISPR-Cas adaptive immunity system has emerged as one of the most robust and precise genetic manipulation tools ever discovered in the field of molecular life s ciences[1,2]
The availability of a curated structure-seq profile with comprehensive structural information made the XIST transcript a potential candidate for testing our hypothesis
The plasmid library was constructed by cloning crRNAs in the pRMT vector containing human optimized LshCas13a insert, followed by transfection in the HEK293T cells and transcript quantification by exon specific primers
Summary
Bacterial CRISPR-Cas adaptive immunity system has emerged as one of the most robust and precise genetic manipulation tools ever discovered in the field of molecular life s ciences[1,2]. Recent pursuits in large-scale data mining of the microbial genomes led to the identification of unexplored classes of RNA-targeting CRISPR-Cas systems including C2c1, C2c2, and C2c3. In sheer contrast to Cas[9] endonucleases, the recruitment of crRNA-Cas[13] effector to the target RNA does not induce local melting necessary for strand separation, thereby hampering crRNA binding to the DS r egions[8]. This rationalizes why the knockdown efficiency of Cas[13] is explicit for SS regions but not for substantially basepaired DS regions. Since Cas[13] selectively prefers single-stranded regions in RNA, we present a structure-based gRNA design strategy for exclusively targeting the SS regions by utilizing structural information derived from structureseq data and structure prediction methods
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