Pooled genome-wide CRISPR screening for basal and context-specific fitness gene essentiality in Drosophila cells

Abstract

Genome-wide screens in Drosophila cells have offered numerous insights into gene function, yet a major limitation has been the inability to stably deliver large multiplexed DNA libraries to cultured cells allowing barcoded pooled screens. Here, we developed a site-specific integration strategy for library delivery and performed a genome-wide CRISPR knockout screen in Drosophila S2R+ cells. Under basal growth conditions, 1235 genes were essential for cell fitness at a false-discovery rate of 5%, representing the highest-resolution fitness gene set yet assembled for Drosophila, including 407 genes which likely duplicated along the vertebrate lineage and whose orthologs were underrepresented in human CRISPR screens. We additionally performed context-specific fitness screens for resistance to or synergy with trametinib, a Ras/ERK/ETS inhibitor, or rapamycin, an mTOR inhibitor, and identified key regulators of each pathway. The results present a novel, scalable, and versatile platform for functional genomic screens in invertebrate cells.

Pooled CRISPR Screens in Drosophila Cells

“…Moreover, these data can be used to determine a false discovery rate, as described in Viswanatha et al. (). A single replicate of a genome‐wide screen yields ∼1000 essential genes at a false‐discovery rate of 5%.…”

“…This Basic Protocol also assumes the use of a Cas9 + S2R+ cell line with an attP integration site together with an existing sgRNA library, such as the library described in Viswanatha et al (2018) and available from Addgene (cat. nos.…”

“…at the gene level (see Support Protocol 3 and Viswanatha et al, 2018). Ideally, this screening approach can be used to identify factors conferring either growth advantage (e.g., resistance to a cytotoxin) or growth disadvantage (i.e., genes essential in a particular context such as genotype or treatment with a cytotoxin).…”

Use of the CRISPR‐Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes

“…For all three approaches, starting with a Cas9-positive cell line increases efficiency. The protocols described here are both based on use of the S2R+-MT::Cas9 cell line, which is described in Viswanatha, Li, Hu, & Perrimon (2018) and available from the Drosophila Genomics Resource Center (DGRC #268;https://dgrc.bio.indiana.edu).…”

“…For either protocol, transfection with the donor and sgRNA constructs can be performed by chemical transfection, such as with Qiagen Effectene (Support Protocol 3), or by electroporation, such as with the Lonza Nucleofect system (Support Protocol 4). Moreover, for both approaches, fluorescenceactivated cell sorting (FACS) is used to identify and perform single-cell isolation of putative fluorescent protein-tagged cells, and this can be followed by image analysis to observe GFP or mNeonGreen in the cells, for example using Cell Profiler (Carpenter et al, 2006) and taking advantage of the fact that the S2R+-MT::Cas9 cell line expresses mCherry (Neumuller et al, 2012;Viswanatha et al, 2018). The mCherry signal can be used to identify cells and can be compared with the signal from the knock-in tag.…”