Pooled CRISPR Screens in Drosophila Cells. Academic Article uri icon

Overview

abstract

  • High-throughput screens in Drosophila melanogaster cell lines have led to discovery of conserved gene functions related to signal transduction, host-pathogen interactions, ion transport, and more. CRISPR/Cas9 technology has opened the door to new types of large-scale cell-based screens. Whereas array-format screens require liquid handling automation and assay miniaturization, pooled-format screens, in which reagents are introduced at random and in bulk, can be done in a standard lab setting. We provide a detailed protocol for conducting and evaluating genome-wide CRISPR single guide RNA (sgRNA) pooled screens in Drosophila S2R+ cultured cells. Specifically, we provide step-by-step instructions for library design and production, optimization of cytotoxin-based selection assays, genome-scale screening, and data analysis. This type of project takes ∼3 months to complete. Results can be used in follow-up studies performed in vivo in Drosophila, mammalian cells, and/or other systems. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Pooled-format screening with Cas9-expressing Drosophila S2R+ cells in the presence of cytotoxin Support Protocol 1: Optimization of cytotoxin concentration for Drosophila cell screening Support Protocol 2: CRISPR sgRNA library design and production for Drosophila cell screening Support Protocol 3: Barcode deconvolution and analysis of screening data.

publication date

  • December 1, 2019

Research

keywords

  • CRISPR-Cas Systems
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • High-Throughput Screening Assays
  • RNA, Guide, Kinetoplastida

Identity

PubMed Central ID

  • PMC6961806

Scopus Document Identifier

  • 85075569372

Digital Object Identifier (DOI)

  • 10.1073/pnas.0603161103

PubMed ID

  • 31763777

Additional Document Info

volume

  • 129

issue

  • 1