(a) Schematic for Gal4/VP16 reporter integration to tag a deletion allele of rb1 exons 2–4 (top) and rb1 exons 2–25 (bottom). Arrowheads designate CRISPR/Cas9 DSBs. CRISPR sgRNAs in two exons are expected to excise the intervening genomic DNA. The targeting vector contains a 5’ homology arm flanking the upstream exon target site and a 3’ homology arm flanking the downstream exon target site. (b, b’) Live confocal image of F0 Tg(UAS:mRFP)tpl2 embryo after 2A-Gal4/VP16 deletion tagging at rb1 exons 2–4. (c, c’) Live confocal image of F1 Tg(rb1-e2-2A-Gal4/VP16) embryo from a founder targeted at rb1 exons 2–25. A deletion from exon 2–25 was not observed in the F1 generation, but the 5’ junction was in frame. (d) Schematic for 2A-Gal4/VP16 deletion tagging of msna exons 2–6. (e, e’) Live confocal image of F0 Tg(UAS:mRFP)tpl2 embryo after 2A-Gal4/VP16 deletion tagging at msna exons 2–6. (f) Somatic reporter efficiency of targeted deletion tagging using 48 bp homology arms for rb1 exons 2–4, rb1 exons 2–25, and msna exons 2–6. Data represents mean +/- s.e.m. of 4 (rb1) and 5 (msna) independent targeting experiments. Scale bars 200 μm (b, c, c’, e); 100 μm (b’, e’).
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