CRISPR/Cas-based tools for the targeted control of plant viruses

Abstract

Plant viruses are known to infect most economically important crops and pose a major threat to global food security. Currently, few resistant host phenotypes have been delineated, and while chemicals are used for crop protection against insect pests and bacterial or fungal diseases, these are inefficient against viral diseases. Genetic engineering emerged as a way of modifying the plant genome by introducing functional genes in plants to improve crop productivity under adverse environmental conditions. Recently, new breeding technologies, and in particular the exciting CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) technology, was shown to be a powerful alternative to engineer resistance against plant viruses, thus has great potential for reducing crop losses and improving plant productivity to directly contribute to food security. Indeed, it could circumvent the "Genetic modification" issues because it allows for genome editing without the integration of foreign DNA or RNA into the genome of the host plant, and it is simpler and more versatile than other new breeding technologies. In this review, we describe the predominant features of the major CRISPR/Cas systems and outline strategies for the delivery of CRISPR/Cas reagents to plant cells. We also provide an overview of recent advances that have engineered CRISPR/Cas-based resistance against DNA and RNA viruses in plants through the targeted manipulation of either the viral genome or susceptibility factors of the host plant genome. Finally, we provide insight into the limitations and challenges that CRISPR/Cas technology currently faces and discuss a few alternative applications of the technology in virus research.

Keywords: CRISPR/Cas; crop; genome editing; plant viruses.

FIGURE 1

Repair pathways for nuclease‐induced double‐stranded breaks. Nonhomologous end‐joining leads to the introduction of random indel (insertion/deletion) mutations, whereas homology‐directed repair can introduce point mutations or sequence insertions through recombination using a donor template. This figure was created using Biorender.

FIGURE 2

A schematic comparison of the class 2 CRISPR/Cas systems. (a) Cas9 represents a type II system and is guided by an sgRNA encoding a spacer bound to a dsDNA target adjacent to a PAM. The HNH and RuvC nuclease domains are activated when the correct base‐pairing occurs and cleave both DNA strands. (b) Cas12a represents a type V system and binds to the DNA sequence complementary to the single crRNA spacer and adjacent to a PAM. The RuvC nuclease domain is activated when the correct base‐pairing occurs and ssDNase activity cleaves both strands. (c) Cas13 represents a type VI system and binds to a ssRNA sequence complementary to the crRNA spacer. The HEPN domains are activated when the correct base‐pairing occurs for ssRNase activity. This figure was created using Biorender.

FIGURE 3

Schematic diagram of class 2 CRISPR/Cas strategies against viruses and targeting the host genomic DNA. On DNA virus entry into the plant cell, the Cas9/sgRNA complex binds to and cleaves DNA target sites. For RNA viruses or the RNA transcripts of pathogens with DNA genomes, both FnCas9 and Cas13a proteins guided by their cognate sgRNA or crRNA, respectively, have been proven to target and cleave the virus genome or transcripts. Alternatively, host susceptibility factors can be disrupted by CRISPR/Cas9 to perturb viral infection. The plant susceptibility (S) genes can be altered by directly targeting their coding regions or by modifying the promoter region sequences to prevent pathogen‐effector binding. In instances where the outcome of disturbing S genes is not extensively studied, the CRISPR toolkit can be used to introduce resistance (R) genes. Using the cellular homology‐directed repair (HDR) machinery, Cas9 can mediate the insertion of an R gene. To avoid whole‐gene disruption, Cas9 base‐editing technology can be used to make specific mutations that are associated with a disease‐resistant trait. This figure was created using Biorender.