CRISPR/Cas9-based insertion of epitope-tag sequences into the genome of Drosophila S2 cells was performed as previously described (Bottcher et al., 2014). DNA sequences coding for GFP- or FLAG-tags were inserted at the 3’ end of the coding region of the U-shaped or dMi-2 gene locus, leading to expression of C-terminally tagged proteins.

In brief, S2 cells stably expressing the Cas9 nuclease (S2[Cas9] cells) were transfected with double stranded linear DNA constructs (1) encoding for sgRNA and (2) providing a template for homologous recombination (HR). Both of these constructs were generated by PCR using gene specific primers (S4 Table). The sgRNA sequences were designed to target Cas9 as close to the respective STOP codon as possible with respect to the nearest available protospacer adjacent motif (PAM) (targeting sequences: CATTTGAGAAAGCCAGCTG (Ush) and TCGAATAATTCCGGCGTCT (dMi-2)). Homologous recombination templates were amplified from plasmids containing GFP- or FLAG-tag sequences including a STOP codon as well as a resistance marker under control of a copia promoter. This insert was amplified using primers containing 60 bp sequences homologous to regions directly up- and downstream of the original STOP codon. In particular, HR templates for C-terminal tagging of U-shaped were amplified using the following plasmids: pSK23 (GFP-tag & Puromycin resistance marker; Addgene #72851) and pSK25 (2xFLAG-tag & Puromycin resistance marker; Addgene #72853). HR templates for C-terminal tagging of dMi-2 were amplified using the following plasmids: pMH3 (GFP-tag & Blasticidin resistance marker; Addgene #52528) and pMH4 (2xFLAG-tag & Blasticidin resistance marker; Addgene #52529).

To favour double strand break repair by HR, the protein amount of key enzymes involved in non-homologues end joining (NHEJ) and microhomology-mediated end joining (MMEJ) was lowered by transfecting S2[Cas9] cells with 1 μg/ml dsRNA targeting lig4 (NHEJ) and mus308 (MMEJ) transcripts. After three days, cells were transfected with HR and sgRNA templates using FuGENE HD transfection reagent (E2311, Promega). Four days post transfection cells were transferred to medium containing 2 μg/ml Puromycin (540411, Merck) or 10 μg/ml Blasticidin (A11139, Gibco) respectively. Cells were kept under selection for at least 14 days or until non-resistant control cells declined.

To retrieve monoclones, cells were serially diluted in 96 well plates. Monoclones were expanded and screened by PCR on genomic DNA using primers flanking the insertion site.

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