A Comprehensive Toolbox for Genome Editing in Cultured Drosophila melanogaster Cells

Abstract

Custom genome editing has become an essential element of molecular biology. In particular, the generation of fusion constructs with epitope tags or fluorescent proteins at the genomic locus facilitates the analysis of protein expression, localization, and interaction partners at physiologic levels. Following up on our initial publication, we now describe a considerably simplified, more efficient, and readily scalable experimental workflow for PCR-based genome editing in cultured Drosophila melanogaster cells. Our analysis at the act5C locus suggests that PCR-based homology arms of 60 bp are sufficient to reach targeting efficiencies of up to 80% after selection; extension to 80 bp (PCR) or 500 bp (targeting vector) did not further improve the yield. We have expanded our targeting system to N-terminal epitope tags; this also allows the generation of cell populations with heterologous expression control of the tagged locus via the copper-inducible mtnDE promoter. We present detailed, quantitative data on editing efficiencies for several genomic loci that may serve as positive controls or benchmarks in other laboratories. While our first PCR-based editing approach offered only blasticidin-resistance for selection, we now introduce puromycin-resistance as a second, independent selection marker; it is thus possible to edit two loci (e.g., for coimmunoprecipitation) without marker removal. Finally, we describe a modified FLP recombinase expression plasmid that improves the efficiency of marker cassette FLP-out. In summary, our technique and reagents enable a flexible, robust, and cloning-free genome editing approach that can be parallelized for scale-up.

An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms

“…Given the dynamic localization of Polo protein during mitosis (Llamazares et al, 1991) we recorded the Polo subcellular localization pattern in live cells through mitosis. Time-lapse confocal imaging of Polo-GFP showed that the protein is localized to centrosomes, spindle, and midbody during cell division, in agreement with the data obtained by immunofluorescence (Llamazares et al, 1991) Previously, PCR generated double-stranded constructs containing GFP and an antibiotic resistance gene have been used for homologous recombination in S2 cells (Bottcher et al, 2014;Kunzelmann et al, 2016). However, selection with a drug resistance gene was used to enrich the population and GFP integration frequency was about 2% as judged by FACS.…”

Reversible perturbations of gene regulation after genome editing in Drosophila cells

“…After enrichment of positive cells by antibiotic selection, the resistance marker can be removed via the Flp/FRT system [12, 13]. In order to study the potential of modified loci to trigger siRNA generation, we introduced a C-terminal GFP-tag at the act5C and rtf1 loci in S2 cells.…”

“…CRISPR/ cas9 -mediated genome editing with 60 nt homology PCR products was performed as previously described [12, 13]. Primer sequences are provided in S1 Table.…”

Generating and working with Drosophila cell cultures: Current challenges and opportunities

“…CRISPR‐Cas9 has been used to edit the genome of at least three Drosophila cell lines (Bassett, Tibbit, Ponting, & Liu, ; Bottcher et al, ; Franz, Shlyueva, et al, ; Ishizu, Sumiyoshi, & Siomi, ; Kunzelmann, Bottcher, Schmidts, & Forstemann, ; S. E. Mohr et al, ). The list of genome‐edited Drosophila cell lines is likely to grow rapidly.…”