Conferring Resistance to Plant RNA Viruses with the CRISPR/CasRx System

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) is an adaptive immune system discovered in prokaryotic bacteria or archaea that can fend off invading nucleic acids. Because of its simplicity, high efficiency and versatility, CRISPR/Cas system-mediated genome editing has been widely applied in plant research and agricultural production. In brief, an effective CRISPR/Cas system contains an easily-engineered guide RNA (gRNA) and a Cas effector protein. Recently, a special type of RNA targeting CRISPR/Cas-associated protein, Cas13a (formerly called C2c2), was found (Shmakov et al. 2015). This new type of Cas protein, unlike Cas9 or Cas12a, can target and edit single-stranded RNA (ssRNA) rather than double-stranded DNA (dsDNA) in vivo (Abudayyeh et al. 2017; Cox et al. 2017). Plant viruses, of which RNA viruses constitute the largest group, are obligate invading parasites that cause devastating diseases in economically important crops. There exist several RNA-targeted anti-viral immunity systems in plants, including RNA silencing, RNA decay and other RNA quality control systems (Li et al. 2019), some of which have been applied into the prevention and control of plant RNA viruses in the field, such as the effective RNA silencing-based antiviral engineering. As the development of CRISPR/Cas systems, these systems-mediated DNA or RNA editing/ interference in plants make them very attractive tools applicable to inhibit the infection of different plant RNA viruses (Cao et al. 2020). Generally, targeting viral RNA sequences and editing DNA sequences of host susceptibility genes are two main CRISPR/Cas technology-mediated strategies to be used for restriction of viral infection in plants (Cao et al. 2020; Pyott et al. 2020). A plant-codon-optimized version of FnCas9 paired with rgRNA targeting plant ssRNA viruses has been employed to gain resistance to several agricultural important plant RNAviruses (Zhang T et al. 2018). Recently, the use of Cas13a as an effective method to inhibit plant RNA virus infection was also demonstrated (Zhang T et al. 2018). Eukaryotic translation initiation factors eIF4E, eIF4G and their isoforms eIF(iso)4E and eIF(iso)4G are the most widely exploited host susceptibility genes edited by CRISPR/Cas9 systems to confer resistance to plant RNA viruses (Chandrasekaran et al. 2016; Cao et al. 2020). A recent work characterizing the functionality of Cas13d from Ruminococcus flavefaciens (CasRx) has shown that the single effector CasRx protein is a programmable RNA-guided ssRNA nuclease. CasRx possesses two higher eukaryotic and prokaryotic nucleotide binding domains (HEPN) that dictate CRISPR RNA maturation and target cleavage (Konermann et al. 2018; Zhang C et al. 2018; Zhang B et al. 2019). CasRx is smaller than Cas13a, Cas13b and other Cas13 variants, and it is effective to combat RNA virus (Mahas et al. 2019). In addition, CasRx is independent of protospacer flanking site (PFS), and HEPN-dependent ribonuclease with RNA cleavage activity (Konermann et al. 2018; Wessels et al. 2020). In this case, besides cleaving the target RNA, CasRx also shows promiscuous ability of the RNase to cleave collateral RNAs once activated in the presence of template targets. Therefore, CasRx-mediated RNA-targeting tools show promise for applications such as RNA virus suppression, gene knockdown, RNA detection, or transcript tracking. Here, we systemically analyzed the effect of CasRx-mediated RNA interference in Nicotiana benthamiana plants defending against exogenous RNAs expressed from a plant virus or from a transiently transformed vector. Supplementary information The online version contains supplementary material available at (https://doi.org/10.1007/s12250020-00338-8).