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

Abstract

The Transgenic RNAi Project (TRiP), a Drosophila functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNAi fly stocks. To date, the TRiP 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 sgRNAs targeting upstream of a gene transcription start site. Gene activation is triggered by co-expression of catalytically dead Cas9 (dCas9) fused to an activator domain, either VP64-p65-Rta (VPR) or Synergistic Activation Mediator (SAM). TRiP-KO stocks express one or two sgRNAs targeting the coding sequence of a gene or genes, allowing for generation of indels in both germline and somatic tissue. To date, we have generated more than 5,000 CRISPR-OE or -KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.

Spatiotemporal recruitment of RhoGTPase protein GRAF inhibits actomyosin ring constriction in Drosophila cellularization

“…Flies containing nos-Cas9 were crossed to ubiquitous expressing gRNA against the first exon of GRAF to obtain F1 males containing nos-Cas9/GRAF gRNA (Ren et al, 2013;Zirin et al, 2020). In the germline of F1 males, the Cas9 enzyme cleaved the first exon of GRAF leading to NHEJ repair and the occurrence of different mutations (Figure 1figure supplement 2B,C).…”

“…In addition, loss of function mutants at the Graf locus were generated by the Crispr-Cas9 strategy. The transgenic fly stock containing a guide RNA (gRNA) against the exon1 (at 2751 residue from transcription start site) of Graf was crossed to maternally expressing Cas9 (nanos-Cas9) (Ren et al, 2013;Zirin et al, 2020). A single gRNA against exon1 of Graf was used to avoid perturbing another putative gene CG8960 present in the intronic region of Graf in the reverse direction.…”

The function of α-Catenin mechanosensing in tissue morphogenesis

“…α-Cat-RNAi (TRiP HMS00317); jub-RNAi (TRiP HMS00714); canoe-RNAi (TRiP HMS00239 and GL00633); Rap1-RNAi (TRiP HMJ21898); were produced by the Transgenic RNAi Project at Harvard (Zirin et al, 2020). GFP-RNAi (BL41552, Perrimon, N.).…”

The RNA-binding protein Musashi controls axon compartment-specific synaptic connectivity through ptp69D mRNA poly(A)-tailing

“…The following references appear in the Supplemental information: Desai et al (1994); Desai et al (1996); Desai and purdy, (2003); Jan and Jan (1982); Morrison and Halder (2010); Parks et al (2004); Zirin et al (2020).…”