Donor transgenic construct for use in the homology assisted CRISPR knock-in (HACK) method. Designed to convert a GAL4 line to a lexA::GAD line in vivo. The construct contains the DNA of interest for incorporation into the genome (in this case a T2A-lexA::GAD cassette, plus an excisable loxP cassette containing a Disc\RFP3xP3.cUa marker) flanked by 5' and 3' homology arms to the targeted sequence which is to be replaced (in this case GAL4). It also contains two tandem guide RNAs (gRNAs) that target the sequence to be replaced; these gRNAs are not included in the converted target. CRISPR/Cas9 is used to induce a double-strand DNA break in the target GAL4 sequence, which is then repaired using the donor as a template, resulting in insertion of the T2A-lexA::GAD cassette and excisable Disc\RFP3xP3.cUa marker in place of the targeted GAL4 sequence. The T2A ribosomal skipping sequence allows separate translation of the remaining GAL4 sequence (a 5' truncated portion) and lexA::GAD sequence from the single transcript that is produced by the converted transgene.
