Background

Currently, most of the genes in Drosophila can be knocked down or knocked out through powerful genetic tools and several loss-of-function (LOF) resources have been established based on these tools (Ren et al., 2013; Qiao et al., 2018). A similar gain-of-function (GOF) resource is valuable to study gene function as it can be a complement to LOF. However, we do not have a genome-wide GOF resource until now mainly because of technical difficulties. The traditional gene overexpression system in Drosophila is the Gal4/UAS:cDNA system. It needs to clone the coding DNA sequence (CDS) of a gene downstream of the upstream activation sequence (UAS) which can be controlled by Gal4 transcription factor. It is laborious, expensive and sometimes unachievable. Therefore, an easier way to realize GOF is urgently needed. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is an effective genome-editing tool. The transcriptional activation system in Drosophila based on the CRISPR/Cas9 technique, termed “CRISPR transcriptional activation (CRISPRa) system”, has been developed in recent years (Konermann et al., 2015; Lin et al., 2015; Ewen-Campen et al., 2017). Cas9 has HNH and RuvC nuclease domains which are responsible for the cutting of complementary and non-complementary DNA strand, respectively. Through introducing single amino acid mutation within HNH and RuvC domains of Cas9 (dCas9), nuclease activity of Cas9 is abolished without affecting its DNA binding ability (Dominguez et al., 2016). The CRISPR transcriptional activation CRISPRa system is generated by fusing several activation domains to dCas9 and can be recruited to the upstream of the Transcription Start Site (TSS) through single-guide RNAs (sgRNAs). The specificity of CRISPRa system is controlled by 20 bp guide sequence in sgRNA, thus one can simply use the 20 bp guide sequence against different targets. Previously, VPR-based CRISPRa system has been built in Drosophila in vivo and in vitro (Lin et al., 2015; Ewen-Campen et al., 2017). In this system, three activation domains, VP64, P65, Rta (VPR), tandemly following the dCas9. However, due to low efficiency, this system relies on two sgRNAs to boost gene transcription, which not only increased the complicacy and cost of the cloning procedure, but also increased toxicity and the chances of off-target effects. In addition, although it works both in vitro and in vivo, it often produces weaker phenotypes than the traditional Gal4/UAS:cDNA system.

To improve the efficiency, reduce the toxicity, and streamline the cloning procedure, this protocol introduces how to construct transcriptional activation lines based on flySAM system. In this system, the dCas9-VP64 and MCP-p65-HSF1 is controlled by 10x UAS and sgRNA2.0 which is driven by U6:2 promoter (Jia et al., 2018). Unlike the previous methods, single sgRNA can successfully activate gene transcription. In addition, to study the intrinsic and extrinsic roles of genes with redundant functions or involved in protein complex, we can easily clone multiple sgRNAs to target different genes.