The Cas9(D10A) entry in FlyBase represents a RNA-guided nuclease that is derived from the naturally occurring Cas9 endonuclease from Streptococcus pyogenes and which contains an inactivating point mutation (D10A) in the RuvC endonuclease domain (the HNH endonuclease domain is not mutated). This converts the endonuclease from one that generates double stranded breaks in DNA to a 'nickase', which cleaves a single DNA strand (PMID:22745249). Cas9(D10A) is used in combination with a chimeric guide RNA (gRNA) composed of a ~20 nucleotide 'spacer' sequence that defines the genomic target site, plus additional sequence required for Cas9(D10A) protein binding (PMID:23287722, PMID:23287718). Provided that a 3 nucleotide protospacer adjacent motif (PAM) sequence (NGG) is present immediately 3' to the 20bp targeted by the gRNA, then a single-stranded DNA cut in the strand complementary to the gRNA can occur (PMID:22745249). If Cas9(D10A) is combined with two offset gRNAs that target neighboring genomic targets on opposite DNA strands (-1 to 26bp apart), it can efficiently produce indel mutations in transgenic flies, with a reduced occurrence of off-target damage compared to that observed using Cas9 and a single gRNA (FBrf0226508, PMID:24584192).