A set of plasmids that each consist of two short guide RNA (sgRNA) sequences that both target the coding region of a gene immediately downstream of the translation initiation site. The sgRNAs are expressed under the control of a ubiquitous promoter. In the presence of a source of Cas9, cleavage and mutagenesis of the targeted gene is induced, resulting in a small deletion or insertion mutation. Each plasmid contains an attB site, allowing integration into an attP-containing docking site in the genome to produce a transgenic construct.