The CRISPR/Cas9 system has emerged as a versatile molecular tool for genome editing in various organisms, including plants. In this system, the specificity of Cas9-directed DNA cleavage strictly requires the presence of a chimeric single guide RNA (sgRNA) and a short trinucleotide proto spacer adjacent motif (PAM) in the genome.

To date, the Cas9 used in plants has only been shown to recognize PAM sequences in the canonical form NGG. As such, the range of sequences for genome editing in plants is limited to sites containing an NGG motif. Recent work has revealed that Cas9 can be modified to recognize alternative PAM sequences in zebrafish and human cells. However, the targeting range limitations in plants have yet to be resolved.

Recently, researchers from China National Rice Research Institute (CNRRI) of Chinese Academy of Agricultural Sciences (CAAS) obtained that two modified SpCas9 variants showed robust genome editing activities at endogenous rice target sites harboring either an NGA or an NGCG PAM, which significantly broadened the range of genome editing. They also detected off-target events in transgenic plants. These results suggest that the specificities of the variants are comparable to that of wild-type Cas9. Taking these findings together, the variants generated in this study enable genome editing in a wider range of plants and could therefore be applied in both basic research and crop breeding in the future.