Targeted gene disruption by CRISPR/xCas9 system in Drosophila melanogaster

Although the Cas9 protein from Streptococcus pyogenes (SpCas9) is the most widely used clustered regularly interspaced short palindromic repeats (CRISPR) variant in genome engineering experiments, it does have certain limitations. First, the stringent requirement for the protospacer adjacent motif (PAM) sequence limits the target DNA that can be manipulated using this method in insects. Second, its complementarity specifications are not very stringent, meaning that it can sometimes cause off-target effects at the target site. A recent study reported that an evolved SpCas9 variant, xCas9(3.7), with preference for various 5 '-NG-3 ' PAM sequences not only has the broadest PAM compatibility but also has much greater DNA specificity and lower genome-wide off-target activity than SpCas9 in mammalian cells. Here we applied the CRISPR/xCas9 system to target the white gene in Drosophila melanogaster, testing the genome-editing efficiency of xCas9 at different PAM sites. On the GGG PAM site, xCas9 showed less activity than SpCas9. For the non-NGG PAM site TGA, xCas9 could produce DNA cleavage and indel-mediated disruption on the target gene. However, for other non-NGG PAM sites, xCas9 showed no activity. These findings show that the evolved Cas9 variant with broad PAM compatibility is functional in Drosophila to induce heritable gene alterations, increasing the targeting range for the applications of genome editing in insects.