Systemic evaluation of various CRISPR/Cas13 orthologs for knock-down of targeted transcripts in plants

CRISPR/Cas13 system, recognized for its compact size and specificity in targeting RNA, has recently been employed for RNA degradation. However, the potential of various CRISPR/Cas13 subtypes, particularly concerning the knockdown of endogenous transcripts, has not yet been comprehensively characterized in plants. In this study, we present a full spectrum of editing profiles for seven Cas13 orthologs from five distinct subtypes: VI-A (LwaCas13a), VI-B (PbuCas13b), VI-D (RfxCas13d), VI-X (Cas13x.1 and Cas13x.2), and VI-Y (Cas13y.1 and Cas13y.2). A systematic evaluation of the knockdown effects on two endogenous transcripts (GhCLA and GhPGF in cotton) as well as an RNA virus (TMV in tobacco) revealed that RfxCas13d, Cas13x.1, and Cas13x.2 exhibited enhanced stability with editing efficiencies ranging from 58% to 80%, closely followed by Cas13y.1 and Cas13y.2. LwaCas13a displayed the lowest level of mRNA cleavage activity, while PbuCas13b demonstrated slightly higher activity compared to LwaCas13a. Both single crRNA and dual crRNAs strategies facilitated efficient knockdown of the same transcript. Notably, both Cas13x.1 and Cas13y.1 can simultaneously degrade two endogenous transcripts through a tRNA-crRNA cassette approach, achieving high editing efficiencies of up to 50%. Furthermore, Cas13 orthologs enable varying degrees of endogenous transcript knockdown with minimal off-target effects, generating germplasms that exhibit a diverse spectrum of mutant phenotypes. Transgenic tobacco plants showed significant reductions in damage along with mild oxidative stress and minimal accumulation of viral particles after TMV infection. In conclusion, our study presents an efficient and robust platform for transcriptome editing that holds promise for plant functional research and future crop improvement.