Implementation of CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing

Abstract

In contrast to genome editing that introduces genetic changes at DNA level, disrupting or editing genes transcripts provides a distinctive approach to perturb a genetic system, offering benefits complementary to classic genetic approaches. To develop a new toolset for manipulation of RNA, we first implemented a member of type VI CRISPR systems, Cas13a from Leptotrichia shahii (LshCas13a) in Schizosaccharomyces pombe, an important model organism employed by biologists to study key cellular mechanisms conserved from yeast to humans. While it was shown to knock down targeted endogenous genes transcripts, differently from previous studies in E. coli, no collateral cleavage of other non-specific RNA by activated Cas13a-crRNA complex was detected in fission yeast. Second, we engineered a RNA-editing system by tethering an inactive form of LshCas13a (dCas13) to the catalytic domain of human Adenosine Deaminase Act on RNA 2 (hADAR2d), which was shown to be programmable with crRNA to target messenger RNAs and precisely edit specific nucleotide residues. We optimized the system parameters using a dual-florescence reporter and demonstrated its utility in editing of randomly selected endogenous genes transcripts. Our engineered RNA-editing system enables a new toolset for transcriptomic manipulation that is widely applicable in basic genetic and biotechnological research.