A CRISPR-dCas13 RNA-editing tool to study alternative splicing

Abstract: Alternative splicing is an RNA processing used by the cell to increase its protein diversity and genome plasticity through generation of several transcripts from the same gene. It affects the vast majority of biological processes, from stem cell differentiation to cell metabolism. However, tools to properly study the role of a specific splice variant are still missing. With the discovery of the bacterial CRISPR system, a new era in nucleic acid editing has emerged. RNA-directed CRISPR/Cas13 RNAses were recently shown to efficiently target the RNA with higher specificity than Cas9 to the DNA. In this work, we are taking advantage of the catalytic dead mutant dCas13 family member dCasRx to edit alternative splicing patterns in a physiological context. Thanks to our new strategy, isoform-switching splicing changes are easily obtained at endogenous genes without impacting overall gene expression levels. Moreover, we propose a new application for this dCasRx splicing editing system to identify the key regulatory elements involved in the alternative splicing of a given gene. This new approach will increase the RNA toolkit to properly understand the biological impact and regulatory mechanisms of alternative splicing in a given biological process or pathological scenario.