The FokI::dCas9 entry in FlyBase represents an artificial RNA-guided nuclease generated by fusing the nuclease domain of the FokI restriction endonuclease to 'dCas9', a catalytically dead form of the Cas9 endonuclease from Streptococcus pyogenes (FokI sequence is N-terminal). DNA cleavage is mediated by the FokI nuclease domain, while binding specificity is mediated by the dCas9 sequence, which requires a guide RNA (gRNA) to target it to a genomic target site of interest. The gRNA is composed of a ~20 nucleotide 'spacer' sequence that defines the genomic target site, plus additional sequence required for dCas9 protein binding (PMID:23287722, PMID:23287718). An additional requirement is the presence of a 3 nucleotide protospacer adjacent motif (PAM) sequence (NGG) at the genomic target site, immediately 3' to the 20bp targeted by the gRNA (PMID:22745249). DNA cleavage by FokI nuclease domains is strictly dimerization dependent, thus DNA cleavage by FokI::dCas9 requires the use of two gRNAs that each bind to a target 'half-site' which have a defined spacing and orientation, such that a FokI::dCas9 dimer can be formed on the DNA and can generate a double-strand break; the length of the spacer sequence between the two sites is ~15-25bp and the PAM sequences of the half-sites must be distal from the spacer ('PAM-out' orientation). These requirements effectively extend the length of the target site for FokI::dCas9 compared to wild-type Cas9 endonuclease, which is expected to increase the specificity of DNA cleavage (PMID:24770325, PMID:24770324).