For genes that permit intronic tagging, we design homology arms within the 5’-most intron that can be used to tag all or most of the predicted splice isoforms. These homology arms are cloned into a donor vector such that they flank a mutagenic CRISPR MiMIC (CRIMIC) gene trap, containing a splice acceptor, stop codons, as well as a T2A-Gal4. Injection of the construct and an sgRNA targeting the intron into Cas9-expressing fly embryos induces a DNA double-strand break in the germline, which allows for integration of the CRIMIC trap by homology directed repair. Once integrated, the CRIMIC insert produces a truncated mRNA of the target gene, as well as Gal4 under the control of the endogenous gene regulatory elements. These cassettes can then be converted into protein traps using established RMCE methods.
Before submitting your nomination, please check the MiMIC database to verify if your gene does not already have a T2A-Gal4 line or a MiMIC insertion suitable for Recombination Mediated Cassette Exchange (RMCE).