Pooled CRISPR Screens in Drosophila Cells.

Area of Science:

• * Genomics and Molecular Biology
• * Cell Biology and Genetics

Background:

• * High-throughput screens in Drosophila melanogaster cell lines have identified conserved gene functions in critical biological processes.
• * CRISPR/Cas9 technology facilitates novel large-scale cell-based screening approaches.
• * Pooled-format screens offer an alternative to array-format screens, requiring less specialized equipment.

Purpose of the Study:

• * To provide a comprehensive protocol for performing and evaluating genome-wide CRISPR single guide RNA (sgRNA) pooled screens in Drosophila S2R+ cells.
• * To outline methods for library design, production, assay optimization, screening, and data analysis.
• * To establish a feasible workflow for functional genomics in Drosophila.

Main Methods:

• * Development and implementation of a pooled-format CRISPR screening strategy using Cas9-expressing Drosophila S2R+ cells.
• * Optimization of cytotoxin-based selection assays for effective screening.
• * Genome-scale screening, including CRISPR sgRNA library design, production, and barcode deconvolution for data analysis.

Main Results:

• * A detailed, step-by-step protocol for conducting genome-wide CRISPR pooled screens in Drosophila S2R+ cells.
• * Demonstration of the feasibility of pooled screening in a standard laboratory setting.
• * Establishment of a ~3-month workflow for completing such screening projects.

Conclusions:

• * Genome-wide CRISPR pooled screens in Drosophila S2R+ cells are a powerful tool for discovering conserved gene functions.
• * The provided protocol enables efficient and accessible large-scale functional genomics studies.
• * Results from these screens can inform follow-up studies in various model systems, including in vivo Drosophila and mammalian cells.