Large-Scale Transgenic Drosophila Resource Collections for Loss- and Gain-of-Function Studies

Abstract

The Transgenic RNAi Project (TRiP), a Drosophila melanogaster functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNA interference (RNAi) fly stocks. To date, it has generated >15,000 RNAi fly stocks. As this covers most Drosophila genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express single guide RNAs targeting upstream of a gene transcription start site. Gene activation is triggered by coexpression of catalytically dead Cas9 fused to an activator domain, either VP64-p65-Rta or Synergistic Activation Mediator. TRiP-KO stocks express one or two single guide RNAs targeting the coding sequence of a gene or genes. Cutting is triggered by coexpression of Cas9, allowing for generation of indels in both germline and somatic tissue. To date, we have generated >5000 TRiP-OE or TRiP-KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.

A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila

“…CRISPR-based techniques are remarkably precise and, therefore, ideal for replacing, validating, and complementing traditional approaches, in particular procedures relying on the expression of RNAi or cDNA transgenes [40,41]. Also, the large worldwide collection of gRNAs stocks has ensured the quick adaptation of CRIS PR/Cas9 into mainstream Drosophila research [6,42,43]. Given the potential of CRIS PR/Cas13-based methods to replace current techniques, we explored its feasibility and reliability in Drosophila.…”

Strategies for Functional Interrogation of Big Cancer Data Using Drosophila Cancer Models

“…Large transgenic Drosophila collections for gene overexpression and knock-down [32][33][34][35] provide an opportunity to explore the biological relevance of large numbers of gene expression changes reported in human tumors. Drosophila orthologs of genes upregulated and downregulated in tumors can be overexpressed and knocked-down, respectively, in Drosophila cancer models in genetic modifier screens.…”

PRPS-Associated Disorders and the Drosophila Model of Arts Syndrome

“…Moreover, libraries containing guide RNA (gRNA)-expressing plasmids and fly lines are being generated, which will allow genetic screening via the CRISPR/Cas9 system [ 44 , 45 ]. Notably, variant forms of Cas9 nucleases are also used in Drosophila that allow overexpression of a gene of interest from its endogenous promoter [ 46 ] These techniques will further improve Drosophila as a model organism to probe and interrogate gene function.…”