Next generation CRISPR/Cas9 transcriptional activation inDrosophilausing flySAM

Abstract

CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic over-expression genetic analysis in Drosophila melanogaster. We present flySAM, a potent new tool for in vivo CRISPRa, which offers a major improvement 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 over-expression. Further, because flySAM typically only requires 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 usage 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 thus replace previous CRISPRa strategies as the basis of our growing genome-wide transgenic over-expression resource, TRiP-OE.

Enhanced Efficiency of flySAM by Optimization of sgRNA Parameters inDrosophila

“…All the sgRNA sequences used in generating transgenic CRISPRa stocks are listed in Table S1. After annealing, the sgRNA was cloned into flySAM2.0 vector (JIA et al 2018). Then the constructed vectors were injected into y, sc,v,nanos-integrase;attP40 embryos to screen the transgenic lines following standard procedures (WU et al 2019).…”

“…sgRNA target sites with the PAM sequence were synthesized and annealed then cloned into the SpeI/BamHI-digested (to remove the U6B: sgRNA elements) sgRNA2.0 luciferase reporter vector to form luciferase reporter plasmid (JIA et al 2018). In these plasmids, firefly luciferase gene is under the control of the HSP70b promoter and the sgRNA target site is 255bp (for experiments comparing the effects of T and NT strand) or 53bp (for remaining experiments) upstream from the TSS.…”

“…Previously, we generated a flySAM-based CRISPRa system in Drosophila, by using a method of T2A-mediated multiple protein expression, which includes the Gal4/UAS system-controlled flySAM and U6b promoter-driven sgRNA (JIA et al 2018). We have observed that the flySAM system can significantly activate target genes with only one sgRNA, and that the severity of phenotypes is comparable to the traditional Gal4/UAS overexpression system.…”

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).…”

“…More recently, a fly SAM system was developed to improve the effectiveness, scalability, multiplexity, and simplicity of existing CRISPR activation strategies (Jia et al 2018). Two versions of flySAM, namely, flySAM1.0 and flySAM2.0 were developed.…”