Abstract
BackgroundRNA-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.ResultsHere, 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.ConclusionsCollectively, 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.
Highlights
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
Results CRISPR RNA (crRNA) guides alone, in the absence of Cas13, can elicit target RNA reduction To test the Cas13 system in plants, we synthesized the coding sequence for two Cas13a proteins, termed LbaCas13a and LbuCas13a (Leptotrichia buccalis) for expression in plants. We tested their function in planta by targeting the plant infecting Turnip mosaic virus (TuMV) expressing GFP by co-expressing Cas13, crRNA targeting TuMV, and TuMV expressing GFP in Nicotiana benthamiana leaves using Agrobacterium-mediated transient expression [19, 20]
The Cas13 proteins were expressed with a single-guide crRNA containing antisense sequence to one region of the TuMV genome, a multi-guide crRNA containing sequence against three regions of the genome, or an empty-guide, which contained the direct repeat (DR) crRNA sequence alone (Fig. 1a)
Summary
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. Genome editing technologies such as CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein), CRISPR-Cas, and newly identified systems enable unprecedented opportunities for genome engineering [1,2,3,4]. The derivative Cas protein, termed PRIME-editor, enables more precise editing and overcomes the unintended consequences resulting from the creation of double-strand breaks [7] Despite these technical advances in genome engineering, there remains a potentially fundamental limitation to DNA editing, where the alteration of a gene results in unintended and unpredictable phenotypes. This approach will likely serve a role in future application of genome engineering, but the generation of mosaic genotypes caused by differences in the rate and penetrance of cell-specific editing, especially in polyploid crops, may limit the utility of this approach
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