TI{CRISPaint.T2A-GAL4} represents a DNA segment that has been integrated into the genome by homology-independent insertion using CRISPR/Cas9 in combination with two sgRNAs - one sgRNA directed to a target genomic locus and one frame-specific sgRNA directed to a CRISPaint donor plasmid. In the presence of Cas9, both the genomic target and the CRISPaint donor plasmid are cut (the donor in a frame-specific manner) and the linearized donor is integrated into the target locus by non-homologous end joining. The inserted TI{CRISPaint.T2A-GAL4} DNA is not flanked by transposable element ends and contains a promoterless coding cassette comprised of the T2A peptide, the GAL4 coding sequence and an SV40 polyadenylation signal, followed by a loxP-flanked Disc\RFP^{DsRed.3xP3.cUa} marker. Homology-independent insertion is less precise than homology-directed repair; the donor sequence can insert in either orientation, insertion/deletion mutations may be introduced at the site of insertion, and concatemers of the donor sequence may be inserted. Integration of TI{CRISPaint.T2A-GAL4} into a Drosophila gene of interest can be used to generate loss-of-function alleles. In addition, if the insertion is in the sense orientation with respect to the targeted gene, GAL4 may be expressed under the control of the regulatory sequences of the targeted gene (the presence of the T2A sequence promotes the separate translation of the GAL4 open reading frame). Although it was expected that GAL4 expression would only occur in those cases where an insertion was in-frame with the targeted gene, GAL4 expression has been observed in some sense, but out-of-frame insertions.