Construction of the dlg1[4K] donor plasmid and transgenic line

To introduce a conditionally expressed V5 tag with FRT recombinase target sites into dlg1, we inserted a 3.34 kB sequence supplied by a donor plasmid into the dlg1 locus at a PAM site 12 bp upstream of the most distal stop codon using homology directed repair (HDR) and CRISPR/Cas9 genome editing.^65,68,69 We constructed the dlg1[4K] donor plasmid (Figure 1) by joining five fragments into a minimal (AMP and ORI) plasmid backbone by In Fusion assembly (Takara, no. 639649). Each fragment was amplified using Q5 polymerase (New England Biolabs) with custom primers (IDT, Coralville IA). The primers used are listed in the key resources table. dlg1 DNA was amplified directly from the Vasa-Cas9 injection strain (BL51324) to ensure sequence compatibility. The minimal AMP ORI backbone was derived from pJFRC203-10XUAS-FRT>STOP>FRT-myrsmGFP-cMyc. (RRID: Addgene 63167). This plasmid also supplied the FRT-STOP-FRT cassette. To visibly identify successful transformants, we added a dsRed fluorescent marker derived from pScarlessHD-2xHA-DsRed (a gift from Kate O’Connor-Giles via Addgene (RRID:Addgene_80822). This cassette, flanked by piggyBac inverted repeats, was inserted at a TTAA sequence within the FRT>STOP>FRT cassette and was subsequently removed by crossing transformant lines to a piggyBac transposase source.

Briefly, to assemble the donor plasmid: dlg1 fragment 1 included a 500 bp dlg1 left homology arm with vector compatible sequence at the 5′ end extending to the left-most FRT sequence of the FRT>STOP>FRT cassette. A C nucleotide was inserted to maintain the reading frame after the flip out event. dlg1 fragment 2 extended from this FRT sequence halfway through the STOP cassette into the pScarless sequence at the 5′-most piggyBac inverted repeat. dlg1 fragment 3 extended from the 5′ piggyBac inverted repeat through the DsRed cassette to sequence adjacent to the right-most piggyBac inverted repeat. A synthesized fragment (dlg1 fragment 4, GenScript:RRID:SCR_002891) extends through the right-most piggyBac inverted repeat, the remaining FRT>STOP>FRT cassette and through 12 bp of the dlg1 C-terminal into the 3X V5 epitope tag. The right homology arm was positioned precisely at the Cas9 cut site; which left 12 bp of the dlg1 coding sequence between the 3′ most FRT sequence and the V5 epitope tag, encoding 4 amino acids of the DLG1 carboxy terminus (KESL). dlg1 fragment 4 also spans the PAM site and a G to A mutation was engineered that mutated the PAM site while maintaining the lysine residue. Lastly, dlg1 fragment 5 extended from the V5 sequence through a 500 bp right homology arm with primer sequence compatible with the vector to complete the donor plasmid. Intermediate cloning steps and the final donor plasmid were sequence verified (GeneWiz, South Plainfield NJ). Donor construct sequence is available upon request.

To perform CRISPR/Cas9 at the dlg1 locus, vasa-Cas9 embryos (BDSC 51324) were injected (BestGene, Chino Hills CA) with the donor plasmid and a guide RNA (gRNA) plasmid; the gRNA was made by annealing sense and antisense oligos homologous to sequence adjacent to and 5′ of the targeted dlg1 PAM site (see key resources table) and cloned into pU6-BbsI-chiRNA.⁶⁵ Transformant lines expressing 3xP3 DsRed in the eye were identified visually and then verified for correct integration into dlg1 by amplifying and sequencing genomic DNA spanning the homology arm breakpoints. This verified that the knocked-in sequence was at the predicted position with no genomic rearrangements or nucleotide substitutions. Transgenic knock-in lines received from BestGene were crossed to Herm{3xP3-ECFP, α-tub-piggyBacK10}M6 (BL55804) to excise the DsRed cassette. DsRed negative progeny were balanced and sequence verified to demonstrate precise excision of the scarless cassette. A single line (dlg1[4K]) was chosen for all subsequent experiments.