The CRISPR/Cas revolution reaches the RNA world: Cas13, a new Swiss Army knife for plant biologists

Abstract

Application of the bacterial CRISPR/Cas systems to eukaryotes is revolutionizing biology. Cas9 and Cas12 (previously called Cpf1) are widely used as DNA nucleases for inducing site-specific DNA breaks for different kinds of genome engineering applications, and in their mutated forms as DNA-binding proteins to modify gene expression. Moreover, histone modifications, as well as cytosine methylation or base editing, were achieved with these systems in plants. Recently, with the discovery of the nuclease Cas13a (previously called C2c2), molecular biologists have obtained a system that enables sequence-specific cleavage of single-stranded RNA molecules. The latest experiments with this and also the alternative Cas13b system demonstrate that these proteins can be used in a similar manner in eukaryotes for RNA manipulation as Cas9 and Cas12 for DNA manipulations. The first application of Cas13a for post-transcriptional regulation of gene expression in plants has been reported. Recent results show that the system is also applicable for combating viral infection in plants. As single-stranded RNA viruses are by far the most abundant class of viruses in plants, the application of this system is of special promise for crops. More interesting applications are imminent for plant biologists, with nuclease dead versions of Cas13 enabling the ability to visualize RNA molecules in vivo, as well as to edit different kinds of RNA molecules at specific bases by deamination or to modify them by conjugation. Moreover, by combining DNA- and RNA-directed systems, the most complex of changes in plant metabolism might be achievable.

RNA-Sequencing Analyses of Small Bacterial RNAs and their Emergence as Virulence Factors in Host-Pathogen Interactions

“…From another perspective, the crucial role of sRNAs is also illustrated through the genetic approach of RNA interference (RNAi) and the large diversity of genome-scale editing tools (for reviews [44,45]), such as Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system. Originally a natural system dedicated to genome editing, as part of the bacterial adaptive immune response system, CRISPR has become the swiss army knife [46] of genetics, with promises and challenges [47] in therapy and biotechnology.…”

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

“…Cas13a also differs in the location of the catalytic sites; in an activated state, the HEPN domain is located at the external surface, whereas the nuclease domains of Cas9 and Cas12a are located inside the protein (Liu et al ; Wolter and Puchta ). Like Cas12a, Cas13a can also process the pre‐crRNA by itself and possesses no tracrRNA (East‐Seletsky et al ).…”

“…Post‐transcriptional gene knockdown, instead of transcriptional regulation, can be interesting when only certain splicing variants are to be regulated, as all isoforms are repressed during transcriptional regulation (Mahas et al ). Additionally, gene activity suppression by post‐transcriptional regulation provides a fast, effective and highly specific means of regulation: existing cytoplasmic mRNAs get cleaved, whereas transcriptional regulation suppresses the production of further mRNA generations, while already produced mRNAs remain active (Schindele et al ; Wolter and Puchta ). An applied example of such post‐transcriptional repression was recently shown to successfully combat an RNA virus in plants, using the Cas13a ribonuclease from Leptotrichia shahii (LshCas13a).…”

Genetic improvement of horticultural crops mediated by CRISPR/Cas: a new horizon of possibilities

“…The most recent genome editing tool is the CRISPR/Cas system, identified as part of the immune mechanism against exogenous DNA in bacteria and Archaea. CRISPR/Cas uses small sequences of non-coding RNAs to guide nucleases to cleave a target DNA (Horvath & Barrangou, 2010) or a target RNA, as recently discovered (Wolter & Puchta, 2018). By using RNA as guiding molecules, this technique dispenses the laborious and expensive step of building and optimizing complex proteins (such as ZFN and TALEs) for DNA recognition, representing a more flexible and viable tool for genome manipulation (Song et al, 2016).…”