Next-generation CRISPR/Cas9 transcriptional activation in Drosophila using flySAM

Abstract

CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in We present flySAM, a potent tool for in vivo CRISPRa, which offers major improvements over existing strategies in terms of effectiveness, scalability, and ease of use. flySAM outperforms existing in vivo CRISPRa strategies and approximates phenotypes obtained using traditional Gal4-UAS overexpression. Moreover, because flySAM typically requires only a single sgRNA, it dramatically improves scalability. We use flySAM to demonstrate multiplexed CRISPRa, which has not been previously shown in vivo. In addition, we have simplified the experimental use of flySAM by creating a single vector encoding both the UAS:Cas9-activator and the sgRNA, allowing for inducible CRISPRa in a single genetic cross. flySAM will replace previous CRISPRa strategies as the basis of our growing genome-wide transgenic overexpression resource, TRiP-OE.

An efficient and multiple target transgenic RNAi technique with low toxicity in Drosophila

“…6d , the chm-Tip60-Gcn5 KD clones showed clearly elevated levels of Wg proteins. Meanwhile, we constructed wg transcriptional activation flies using the CRISPRa system 34 , which showed exactly similar eye phenotype as the chm-Tip60-Gcn5 KD when driven by GMR-Gal4 (Fig. 6e ).…”

“…7b ), supporting the specificity of this RNAi experiment. Furthermore, we also generated a transgenic activation line that targeting these three genes simultaneously using the flySAM system we developed recently 34 . As shown in Supplementary Fig.…”

How to study enhancers in non-traditional insect models

“…The initial studies utilizing the dCas9-effector fusion proteins focused on the transcribed regions of the genome [coding regions as well as long non-coding RNA (lncRNA) loci] (e.g. Ewen-Campen et al, 2017;Jia et al, 2018;Lin et al, 2015); however, this technique was later successfully used to modulate enhancer functions (e.g. Thakore et al, 2015, reviewed in Klein et al, 2018Lopes et al, 2016).…”

“…For instance, Xu et al (2017) have used a split-drive configuration of a CRISPR/Cas9based gene drive system (dubbed as CopyCat) to replace the wing vein enhancer of the knirps (kni) gene in Drosophila with that of a mutant allele or the homologous enhancers of other dipteran species. Also, some next-generation CRISPR/Cas technologies, such as CRISPRa, have been successfully used in vivo using Drosophila (Ewen-Campen et al, 2017;Jia et al, 2018;Lin et al, 2015). Although not CRISPR/Cas9-based, Crocker and Stern (2013) used a method that is conceptually similar to dCas9effector technologies (transcription activator-like effectors, TALEs) to interrogate enhancer function in Drosophila, demonstrating that the dCas9-effector technologies can be quite useful when studying insect enhancers.…”

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

“…Target specificity is conferred by 20 bp protospacer sequences in the sgRNA, such that production of reagents for CRISPR activators (CRISPRa) at genome-wide scale is feasible. We reported the first demonstration of effective CRISPR/Cas9-based transcriptional activation in flies , and have since developed and optimized two systems for CRISPRa in vivo (Chavez et al 2016;Jia et al 2018). Based on these methods, we have now generated a collection of transgenic TRiP-CRISPR overexpression (TRiP-OE) lines for gene activation (Fig.…”