Optimized strategy for in vivo Cas9-activation in Drosophila

Abstract

While several large-scale resources are available for in vivo loss-of-function studies in , an analogous resource for overexpressing genes from their endogenous loci does not exist. We describe a strategy for generating such a resource using Cas9 transcriptional activators (CRISPRa). First, we compare a panel of CRISPRa approaches and demonstrate that, for in vivo studies, dCas9-VPR is the most optimal activator. Next, we demonstrate that this approach is scalable and has a high success rate, as>75% of the lines tested activate their target gene. We show that CRISPRa leads to physiologically relevant levels of target gene expression capable of generating strong gain-of-function (GOF) phenotypes in multiple tissues and thus serves as a useful platform for genetic screening. Based on the success of this CRISRPa approach, we are generating a genome-wide collection of flies expressing single-guide RNAs (sgRNAs) for CRISPRa. We also present a collection of more than 30 Gal4 > UAS:dCas9-VPR lines to aid in using these sgRNA lines for GOF studies in vivo.

A method for rapid selection of randomly induced mutations in a gene of interest using CRISPR/Cas9 mediated activation of gene expression

“…Previous work demonstrates CRISPR/Cas9 mediated in vivo activation of hnt expression. In this "proof-of-principle" example, ectopic hnt expression is shown in the third larval instar wing imaginal disc (11). To further investigate CRISPR/Cas9 mediated overexpression of hnt, we used the TRiP-TOE insertion line TOE-GS00052, which ubiquitously expresses two sgRNAs, targeted to 110-129 bps and 183-203 bps upstream of the TSS of hnt (Fig.…”

“…Building on the GAL4/UAS system of inducible gene expression, CRISPR/Cas9 approaches for tissue specific target gene knockdown or overexpression in Drosophila are now also possible; resources, including >1600 transgenic stocks expressing different sgRNAs made by the Drosophila RNAi Screening Center and Transgenic RNAi Project (DRSC/TRiP) are publically available -see (10) for overview of resources. In the case of targeted gene overexpression, the TRiP transgenic lines were designed to ubiquitously express two sgRNAs targeted to sequences upstream of a gene's transcriptional start site (TSS) (11,12). These lines are known as TRiP-OE, or TOE lines.…”

Large-scale transgenicDrosophilaresource collections for loss- and gain-of-function studies

“…Finally, generating a useful genome-wide collection requires that the large majority of the lines generated are functional. To estimate the proportion of TRiP-OE sgRNA lines that function as predicted, we previously created a panel of transgenic fly stocks expressing sgRNAs and analyzed activation using qPCR (Ewen-Campen et al 2017). Seventy-five percent of these sgRNA transgenes led to a greater than three-fold increase in transcript levels when combined with dCas9-VPR, and 58% caused a greater than eight-fold increase, a success rate comparable to the proportion of transgenes in current RNAi stock collections that confer effective knockdown.…”

In vivo epigenome editing and transcriptional modulation using CRISPR technology

“…Using Drosophila models, three independent studies have developed robust in vivo CRISPR activation systems via genetic crosses between dCas9 activator- and sgRNA-expressing flies (Ewen-Campen et al 2017; Jia et al 2018; Lin et al 2015) (Figure 3B). The first demonstration of dCas9-based activation in a multicellular animal was achieved through the genetic crossing of flies with genotype dCas9-VPR and a homozygous sgRNA (Lin et al 2015).…”

“…The Gal4 transcription activator was used to drive expression of the dCas9-VPR by binding to UAS enhancer regions. To enable high throughput and systematic overexpression genetic analysis, a genome-wide collection of flies expressing sgRNAs was subsequently generated for crossing with the dCas9-VPR transgenic flies (Ewen-Campen et al 2017). This approach enabled the generation of easily recognizable gain-of-function phenotypes in multiple tissues in vivo for facilitating large-scale genetic screens.…”