Of particular importance, C2c2-designated as Cas13-has been extensively repurposed in transcriptome engineering for its programmable possibilities in RNA knockdown and RNA editing, transcript tracking, and disease diagnosis 4. These systems comprise of unique interference module distinct from previously characterized DNA targeting systems 3. 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.
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.īacterial 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 sciences 1, 2. 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. We observed that gRNAs targeting the SS regions predicted from the structure, efficiently induced necrosis compared to gRNAs that target the DS regions. 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.
Further, we identified the “central seed region” in the gRNA that upon targeting the SS regions efficiently facilitates Cas13 mediated cleavage. 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. We herein present rational gRNA design by integrating experimental structure-seq data and predicted structural models.
Hence, it becomes indispensable to identify the SS regions for effective Cas13 mediated RNA knockdown. Cas13 endonuclease activity depends on the RNA local secondary structure with strong preference for single-stranded (SS) regions.
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