A Cas9-donor transgenic construct that carries a 2A-Cas9 coding sequence flanked by GAL4 homology arms, a CR7T-U63(2.1) dual-gRNA cassette that ubiquitously expresses 2 gRNAs targeting GAL4 coding sequences (one using a pol III promoter reported to be potent in both the soma and the germline (CR7T) and the other using the snRNA:U6:96Ac promoter), and a ubiquitous nuclear mTagBFP2 marker for separating the Cas9-donor insertion from the converted Cas9. When combined with "nos-Cas9" and the GAL4 line of interest, the gRNAs generate double-strand breaks in the middle of the GAL4 coding sequence and allow in-frame incorporation of 2A-Cas9 via homology-directed repair. The 'self-cleaving' 2A peptide will release Cas9 and a truncated and nonfunctional GAL4 as two separate proteins after translation.