CRISPR guides induce gene silencing in plants in the absence of Cas.

RNA-targeting CRISPR-Cas can provide potential advantages over DNA editing, such as avoiding pleiotropic effects of genome editing, providing precise spatiotemporal regulation, and expanded function including antiviral immunity.Here, we report the use of CRISPR-Cas13 in plants to reduce both viral and endogenous RNA. Unexpectedly, we observe that crRNA designed to guide Cas13 could, in the absence of the Cas13 protein, cause substantial reduction in RNA levels as well. We demonstrate Cas13-independent guide-induced gene silencing (GIGS) in three plant species, including stable transgenic Arabidopsis. Small RNA sequencing during GIGS identifies the production of small RNA that extend beyond the crRNA expressed sequence in samples expressing multi-guide crRNA. Additionally, we demonstrate that mismatches in guide sequences at position 10 and 11 abolish GIGS. Finally, we show that GIGS is elicited by guides that lack the Cas13 direct repeat and can extend to Cas9 designed crRNA of at least 28 base pairs, indicating that GIGS can be elicited through a variety of guide designs and is not dependent on Cas13 crRNA sequences or design.Collectively, our results suggest that GIGS utilizes endogenous RNAi machinery despite the fact that crRNA are unlike canonical triggers of RNAi such as miRNA, hairpins, or long double-stranded RNA. Given similar evidence of Cas13-independent silencing in an insect system, it is likely GIGS is active across many eukaryotes. Our results show that GIGS offers a novel and flexible approach to RNA reduction with potential benefits over existing technologies for crop improvement and functional genomics.

Duke Scholars

Author Jun Huang