Decoding the Mechanism of CRISPR Interference Provided By RNA-Guided RNA-Targeting Cas13a Effector

Type VI-A CRISPR-Cas system relies on Cas13a effector that was shown to target RNA and exhibit a promiscuous RNase activity in vitro, degrading non-specific RNAs upon target recognition. Cas13a significantly decreases cell growth upon targeting a non-essential mRNA, suggesting that RNA-targeting induces cell dormancy and/or programmed cell death [1].

In this work, we studied the cause of growth restriction due to RNA-targeting by Cas13a system from Leptotrichia shahii heterologously expressed in Escherichia coli. Live microscopy revealed that >90% cells expressing Cas13a and RNA target ceased to divide but remained viable. RNA-seq analysis identified ~ 9500 putative Cas13a-dependent cleavage sites in cellular RNA. Bioinformatics analysis suggests that many of these cleavage sites may be secondary effects of induction of cellular RNases belonging to Type II toxin-antitoxin systems. Primer extension assays also validated Cas13a-dependent mRNA and tRNA cleavage products.

Our findings will in turn address the outstanding question of the role of Cas13a as a prokaryotic defense system.

[1] Abudayyeh, O.O., et al., C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science, 2016. 353(6299): p. aaf5573.