Viral diseases are a leading cause of worldwide yield losses in crop production. Breeding of resistance genes (R gene) into elite crop cultivars has been the standard and most cost-effective practice. However, R gene-mediated resistance is limited by the available R genes within genetic resources and in many cases, by strain specificity. Therefore, it is important to generate new and broad-spectrum antiviral strategies. The CRISPR-Cas9 (clustered regularly interspaced palindromic repeat, CRISPR-associated) editing system has been employed to confer resistance to human viruses and several plant single-stranded DNA geminiviruses, pointing out the possible application of the CRISPR-Cas9 system for virus control. Here, we demonstrate that strong viral resistance to cauliflower mosaic virus (CaMV), a pararetrovirus with a double-stranded DNA genome, can be achieved through Cas9-mediated multiplex targeting of the viral coat protein sequence. We further show that small interfering RNAs (siRNA) are produced and mostly map to the 3' end of single-guide RNAs (sgRNA), although very low levels of siRNAs map to the spacer region as well. However, these siRNAs are not responsible for the inhibited CaMV infection because there is no resistance if Cas9 is not present. We have also observed edited viruses in systematically infected leaves in some transgenic plants, with short deletions or insertions consistent with Cas9-induced DNA breaks at the sgRNA target sites in coat protein coding sequence. These edited coat proteins, in most cases, led to earlier translation stop and thus, nonfunctional coat proteins. We also recovered wild-type CP sequence in these infected transgenic plants, suggesting these edited viral genomes were packaged by wild-type coat proteins. Our data demonstrate that the CRISPR-Cas9 system can be used for virus control against plant pararetroviruses with further modifications.
Keywords: CRISPR‐Cas9; cauliflower mosaic virus; small RNA; virus escape; virus resistance.