Tissue-Specific CRISPR-Cas9 Screening in Drosophila

Abstract

Over the last century research in Drosophila has resulted in many fundamental contributions to our understanding of the biology of multicellular organisms. Many of these breakthroughs have been based on the identification of novel gene functions in large-scale genetic screens. However, conventional forward-genetic screens have been limited by the random nature of mutagenesis and difficulties in mapping causal mutations, while reverse-genetic RNAi screens suffer from incomplete knockdown of gene expression. Recently developed large-scale CRISPR-Cas9 libraries promise to address these limitations by allowing the induction of targeted mutations in genes with spatial and temporal control. Here, we provide a guide for tissue-specific CRISPR screening in Drosophila, including the characterization of Gal4 UAS-Cas9 lines, selection of sgRNA libraries, and various quality control measures. We also discuss confounding factors that can give rise to false-positive and false-negative results in such experiments and suggest strategies on how to detect and avoid them. Conditional CRISPR screening represents an exciting new approach for functional genomics in vivo and is set to further expand our knowledge of the molecular underpinning of development, homeostasis, and disease.

Functional septate junctions between cyst cells are required for survival of transit amplifying male germ cells expressing Bag of marbles

“…While we did not detect phenotypes in RNAi crosses for many of the genes that were upregulated as cyst cells differentiate, this could be in part due to failure of the RNAi lines tested to knock down mRNAs strongly enough to reveal gene function (Dietzl et al, 2007; Perkins et al, 2015). Further work using stronger Gal4 drivers and expression systems, loss of function mutations, or tissue-specific CRISPR to induce cell type specific loss of gene function (Port & Boutros, 2022) could lead to the identification of functional roles for additional genes upregulated as cyst stem cells differentiate.…”

Expanding the Drosophila toolkit for dual control of gene expression

“…Most reagents available for loss-of-function (LOF) and gain-of-function studies using RNAi or CRISPR rely on GAL4/UAS-mediated expression ( Brand and Perrimon, 1993 ; Dietzl et al, 2007 ; Perkins et al, 2015 ; Zirin et al, 2020 ; Port and Boutros, 2022 ). However, some studies, such as the study of intercellular or inter-organ communication, require the simultaneous use of two independent binary transcriptional systems.…”

Dissecting neuron-specific functions of circadian genes using modified cell-specific CRISPR approaches

“…Although this approach works well in many cases and has been invaluable for cell-type specific perturbations, it is often variable and usually induces partial knockdown (Dietzl et al, 2007;Meltzer et al, 2019). Fortunately, it is now possible to disrupt genes completely and in the same cell-specific manner using CRISPR/Cas9-based gene editing (Port et al, 2014;Port and Boutros, 2022). Cas9 is an enzyme that induces a double stranded break at a specific genomic location determined by the guideRNA (gRNA) sequence (Jinek et al, 2012).…”

“…The error prone repair machinery endogenous to all cells usually leads to mutations known as indels (insertions-deletions). These range from a missense mutation to a small addition or deletion causing frameshift mutations, often disrupting protein function (Port and Boutros, 2022).…”