The pCFD5 vector is designed to facilitate cloning of one or multiple Cas9-specific guide RNAs (gRNAs) downstream of the strong, ubiquitous U6:3 (snRNA:U6:96Ac) promoter. The gRNAs are flanked by tRNAs and are initially expressed as a single polycistronic tRNA-gRNA cassette; after this primary transcript is expressed, the tRNAs should form secondary structures that are recognized by the endogenous tRNA-processing machinery (RNase enzymes P and Z), resulting in the release of the gRNAs (PMID:25733849). The vector contains an attB site, allowing integration into an attP-containing docking site in the genome to produce a transgenic construct.