Dear Editor, 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 protospacer adjacent motif (PAM) in the genome (Anders et al.,2014;Sternberg et al.,2014).To date,the Cas9 used in plants has only been shown to recognize PAM sequences in the canonical form NGG (Li et al.,2013;Miao et al.,2013;Shan et al.,2013;Ma et al.,2015b).As such,the range of sequences for genome editing in plants is limited to sites containing an NGG motif.Many attempts have been made to overcome this constraint,and recent work has revealed that Cas9 can be modified to recognize alternative PAM sequences in zebrafish and human cells (Kleinstiver et al.,2015).However,the targeting range limitations of the CRISPR/Cas9 systems in plants have yet to be resolved.The widely cultivated plant rice is not only an important food crop but also a model crop plant because of its relatively small genome and relative ease of transformation.To expand the range of genome editing in rice,we generated two Cas9 variants as reported (Kleinstiver et al.,2015) and investigated their genome editing performance in rice.We demonstrate that Cas9 can be engineered to target sites containing alternative non-canonical PAMs in rice,which significantly broadens the range of genome editing.
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