RNA virus interference via CRISPR/Cas13a system in plants

Abstract

BackgroundCRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single-stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants.ResultsCRISPR/Cas13a produces interference against green fluorescent protein (GFP)-expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. CRISPR RNA (crRNAs) targeting the HC-Pro and GFP sequences exhibit better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs.ConclusionsOur data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses and for other RNA manipulations in plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1381-1) contains supplementary material, which is available to authorized users.

RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a

“…Understanding the specificity of Cas13a activator-RNA binding and subsequent HEPN- nuclease activation is crucial for all current (Abudayyeh et al, 2017; Aman et al, 2018; Cox et al, 2017; East-Seletsky et al, 2016; East-Seletsky et al, 2017; Gootenberg et al, 2018; Gootenberg et al, 2017; Konermann et al, 2018) and potential applications of Cas13a’s programmable RNA-binding and/or cleavage capabilities. In particular, recent reports of specific RNA-cleavage in plant (Abudayyeh et al, 2017; Aman et al, 2018) and human (Abudayyeh et al, 2017; Cox et al, 2017; Konermann et al, 2018) cell lines, as well as RNA-editing (Cox et al, 2017) and modulation of pre-mRNA splicing (Konermann et al, 2018) in human cell lines underscore the need to understand how Cas13 is able to specifically bind and in some cases cleave specific RNA targets while avoiding similar sequences, across a range of organisms.…”

“…In particular, recent reports of specific RNA-cleavage in plant (Abudayyeh et al, 2017; Aman et al, 2018) and human (Abudayyeh et al, 2017; Cox et al, 2017; Konermann et al, 2018) cell lines, as well as RNA-editing (Cox et al, 2017) and modulation of pre-mRNA splicing (Konermann et al, 2018) in human cell lines underscore the need to understand how Cas13 is able to specifically bind and in some cases cleave specific RNA targets while avoiding similar sequences, across a range of organisms. Our data enable rational design of crRNAs with optimal specificity and activity for each type of application, and underscore a need to consider the range of effects that Cas13a off-target recognition might have on Cas13a’s ssRNA-binding and/or cleavage behavior, and ultimately the fate of the RNA pool present in each experiment.…”

“…The RNA-activated RNA cleavage behavior of Cas13 provides a mechanism for RNA detection and diagnostic applications (East-Seletsky et al, 2016; Gootenberg et al, 2018; Gootenberg et al, 2017), as well as RNA-targeting in heterologous systems (Abudayyeh et al, 2017; Aman et al, 2018; Cox et al, 2017; Konermann et al, 2018). Versions of Cas13 where the HEPN-domains have been catalytically inactivated (Abudayyeh et al, 2016; East-Seletsky et al, 2016; Konermann et al, 2018; Smargon et al, 2017; Yan et al, 2018) (deactivated Cas13; dCas13) have shown to be useful as programmable RNA binding proteins for RNA imaging (Abudayyeh et al, 2017), RNA editing (Cox et al, 2017) and regulating specific transcripts RNA in other ways (e.g.…”

Disruptive Technology: CRISPR/Cas-Based Tools and Approaches

“…This might partly be addressed by ex vivo selection of genome-edited cells [162], by refinement of additives for the maintenance of stemness in ex vivo procedures and culture [30, 163], by timing DSB induction with suitable phases of the cell cycle, or by improving the selection procedure for enrichment of true stem cells [164], which would also help lower vector requirements and the cost of the procedure. Moreover, Cpf1 appears to have an advantage over Cas9 molecules in general for certain HDR-mediated knock-in or editing events, which might partly relate to the staggered DSB introduced by Cpf1, akin to that produced by TALENs [165, 166]. Additionally, the nature and symmetry of the HDR donor template affect HDR efficiency [166, 167].…”

“…Moreover, Cpf1 appears to have an advantage over Cas9 molecules in general for certain HDR-mediated knock-in or editing events, which might partly relate to the staggered DSB introduced by Cpf1, akin to that produced by TALENs [165, 166]. Additionally, the nature and symmetry of the HDR donor template affect HDR efficiency [166, 167]. Therefore, many factors allow optimization of HDR efficiency, from cell isolation, selection and culture to choice of RGN and donor sequence and type.…”