The Cas9 entry in FlyBase represents a RNA-guided nuclease which is derived from the naturally occurring Cas9 endonuclease from Streptococcus pyogenes and in which the two nuclease domains present in the naturally occurring protein are functional. The exact sequence of the nuclease may differ depending on the particular transgene or modified endogenous locus being used, for example, the coding sequence may have been codon optimized for use in different species. Cas9 nuclease is used in combination with a chimeric guide RNA (gRNA) that contains 20 nucleotides complementary to the genomic target site of interest, plus additional sequence required for Cas9 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 cleavage at the genomic target site can occur. The two nuclease domains present in the Cas9 monomer each cleave one DNA strand (PMID:22745249), resulting in a double stranded break (DSB) which can then be repaired by one of two major DNA repair pathways: non-homologous end joining (NHEJ) or homologous recombination (HR). Since repair by NHEJ can be inaccurate, repair via this pathway typically results in mutations in the genome (insertions or deletions). Repair via HR uses a homologous DNA template, and this property can be exploited to precisely insert or replace defined sequences within the genome (strategies reviewed in FBrf0223980, FBrf0225106, FBrf0228344, FBrf0231034).