“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.

Deep screening of proximal and distal splicing-regulatory elements in a native sequence context

“…Our analysis also suggests that longer gRNA spacers of 25-nt provided a much higher signal-to-noise ratio than shorter gRNA spacers of 22-nt, suggesting that longer spacers help stabilize the gRNA or form a more stable dCas13d/gRNA/target RNA ternary complex. The effect of spacer length on the efficacy of splicing modulation observed at a low MOI with more moderate gRNA expression appears to be clearer than the differences observed upon overexpression of both dCas13d and individual gRNAs 42,45,47,56 .…”

“…Several studies indicated that gRNA length may affect the efficiency of Cas13d-mediated target RNA cleavage 42,45,47,56 . In our initial transfections, we used the 22-nt gRNA D [9,30] targeting the ISS-N1 region as a positive control segRNA.…”

“…In addition, the catalytically dead Cas13 (dCas13) has been used as a programmable RNA-binding module, fused to various effector domains for applications in RNA editing 49 , m 6 A modifications 50 , modulation of splicing 37,51,52 and polyadenylation 53 , live cell RNA imaging 54 , and mapping of protein-RNA interactions through proximity ligation 55 . Finally, attempts to modulate splicing 37,51,56,57 or translation 57 by using dCas13/gRNA to compete with endogenous RNA-binding effectors (e.g., RBP) through steric hindrance have yielded results with varying degree of success. So far, dCas13 applications have been limited to individual gRNAs and targets, with components frequently introduced through transient overexpression.…”

CRISPR-Cas tools for simultaneous transcription & translation control in bacteria

“…In mammalian systems, Cas13 has been used for genome-wide knockdown screens, targeted RNA silencing, and nucleic acid detection (8,10,11). The use of catalytically inactive dCas13 fused to an effector domain has also enabled RNA editing and splicing applications (9,(12)(13)(14). In E. coli, the use of dCas13 for translational repression (CRISPRi) and activation (CRISPRa) of endogenous transcripts has been demonstrated recently (15)(16)(17)(18).…”