CRISPR-Cas13d mediates robust RNA virus interference in plants

Abstract

Background: CRISPR-Cas systems endow bacterial and archaeal species with adaptive immunity mechanisms to fend off invading phages and foreign genetic elements. CRISPR-Cas9 has been harnessed to confer virus interference against DNA viruses in eukaryotes, including plants. In addition, CRISPR-Cas13 systems have been used to target RNA viruses and the transcriptome in mammalian and plant cells. Recently, CRISPR-Cas13a has been shown to confer modest interference against RNA viruses. Here, we characterized a set of different Cas13 variants to identify those with the most efficient, robust, and specific interference activities against RNA viruses in planta using Nicotiana benthamiana.Results: Our data show that LwaCas13a, PspCas13b, and CasRx variants mediate high interference activities against RNA viruses in transient assays. Moreover, CasRx mediated robust interference in both transient and stable overexpression assays when compared to the other variants tested. CasRx targets either one virus alone or two RNA viruses simultaneously, with robust interference efficiencies. In addition, CasRx exhibits strong specificity against the target virus and does not exhibit collateral activity in planta. Conclusions: Our data establish CasRx as the most robust Cas13 variant for RNA virus interference applications in planta and demonstrate its suitability for studying key questions relating to virus biology.

Cas13b-dependent and Cas13b-independent RNA knockdown of viral sequences in mosquito cells following guide RNA expression

“…In this study we have shown that Cas13b RNA guides are able to induce the knockdown of target genes in mosquito cells, both through Cas13b-mediated enhancement of suppression and, unexpectedly, through Cas13b-independent, sequence-specific, RNA knockdown. This is in contrast to work describing the use of Cas13b guides in mammalian and plant cells where RNA knockdown was entirely dependent on the presence of Cas13b 12,17 . We hypothesise that the phenomenon is dependent on base-pair complementarity between the guide and the target RNA, leading to recognition and processing of the target RNA by cellular RNA interference (RNAi) systems.…”

“…The Cas13b-independent effect was detected due to the use of a ZsGreen mock control plasmid (pZsG). Previous work in plant and mammalian cells has not reported interference by the guides alone 12,17 . In plants, the targeting guides were used without Cas13b 17 , while in mammals, the authors used a catalytically inactive dCas13b 12 .…”

“…Previous work in plant and mammalian cells has not reported interference by the guides alone 12,17 . In plants, the targeting guides were used without Cas13b 17 , while in mammals, the authors used a catalytically inactive dCas13b 12 . We speculate that the difference with our results may be due to the cell type used, and that the dCas13b may bind and sequester the guides such that these are less available to endogenous RNAi pathways.…”

“…On the other hand, mutations identified with this split replication system have been shown to have similar effects when introduced into replicating virus 19 . Also, Cas13b-mediated RNA virus inhibition has been demonstrated in both human and plant cells 17,18 .…”

Transcriptome-wide Cas13 guide RNA design for model organisms and viral RNA pathogens

“…Previously, we have described a set of optimal design rules for RfxCas13d guide RNAs (gRNAs), and developed a computational model to predict gRNA efficacy for all human protein-coding genes 5 . However, there is a growing interest to target other types of transcripts, such as noncoding RNAs (ncRNAs) 6,7 or viral RNAs 8,9 , and to target transcripts in other commonly-used organisms [10][11][12][13] . Here, we predicted relative Cas13-driven knock-down for gRNAs targeting messenger RNAs and ncRNAs in six model organisms (human, mouse, zebrafish, fly, nematode and flowering plants) and four abundant RNA virus families (SARS-CoV-2, HIV-1, H1N1 influenza and MERS).…”