Here we describe the TRiP platform for the generation of genome scale collections of Drosophila sgRNA stocks. To search and obtain specific stocks, please visit the reagents page.
TRiP-CRISPR Overexpression (TRiP-OE)
In contrast to the wealth of reagents available for loss-of-function (LOF) studies in Drosophila, the development of large-scale resources for gain-of-function (GOF) studies has lagged behind. This is a major gap in functional genomics, as mis- and overexpression screens are equally informative for elucidating gene functions. The TRiP-OE collection is based on work from the Perrimon lab in which it was demonstrated that CRISPR/Cas9-based transcriptional activation is effective in vivo in Drosophila (Lin et al., 2015).
- Each transgenic TRiP-OE fly stock constitutively expresses two sgRNAs that target ~500 base pairs upstream of the transcriptional start site (TSS) of a single gene of interest
- TRiP-OE stocks are crossed to a stock in which Gal4 directs expression of a catalytically inactive dead Cas9 (dCas9) fused to a highly active chimeric activator called VPR (composed of the VP64, p65, and Rta domains)(Chavez et al., 2015)
- In the resulting progeny (Gal4>dCas9-VPR; sgRNA-gene) the gene of interest is overexpressed in the Gal4 domain
Images courtesy of Ben Ewen-Campen/Perrimon Lab
Examples of TRiP-OE in vivo. Flies homozygous for sgRNA-wg or sgRNA-cut were crossed to flies containing dpp-Gal4 or hh-Gal4 driving expression of UAS-dCas9-VPR activators. In the absence of dCas9-VPR, both genes are expressed in a stripe along the DV wing margin (empty arrowhead). Using dpp-Gal4 (stripe along A-P axis) or hh-Gal4 (posterior of wing), both genes are ectopically activated (white arrowheads). On the right are additional examples of hh-Gal4 driven Cas9-activation phenotypes in adult wings for sgRNA lines targeting scute, CG2889 and CG6066. hh-Gal4 drives expression in the posterior of the wing (below dotted line).
TRiP-CRISPR Knockout (TRiP-KO)
We, and others, have found that the CRISPR/Cas9 system efficiently generates double strand breaks (DSBs) in Drosophila, which can be used effectively to generate mutations or for genome engineering approaches (Bassett et al., 2013; Gratz et al., 2013; Kondo and Ueda, 2013; Ren et al., 2013; Sebo et al., 2014; Yu et al., 2013). TRiP-KO flies ubiquitously express sgRNAs targeting gene coding sequence. Mutant animals or tissue-specific mosaics can be produced by simply crossing TRiP-KO flies to germline-specific-Cas9 or somatic tissue-specific-Gal4>Cas9 flies, respectively (Kondo and Ueda, 2013; Port et al., 2014).
- Each transgenic TRiP-KO fly stock constitutively expresses one sgRNA that targets the coding sequence of a single gene
- To produce mutant animals, TRiP-KO stocks are crossed to flies with a germline-specific source of Cas9 (e.g. nanos-Cas9)
- To produce somatic tissue mutant mosaics, TRiP-KO stocks are crossed to flies with a tissue-specific source of Cas9
- Cas9 induces cleavage of the target gene and mutagenesis via Non-Homologous End Joining (NHEJ) repair
Images courtesy of Perrimon Lab
Examples of TRiP-KO mosaics in vivo. esg-GAL4, tub-GAL80ts, UAS-GFP, UAS-Cas9 flies were crossed to TRiP-KO stocks targeting four different genes with known phenotypes in Drosophila intestinal stem cells (ISCs). In all cases the expected mutant phenotypes were readily detectable and highly penetrant, indicating that the sgRNAs were able to mutate both copies of the genes in most ISCs.
Alejandro Chavez, Jonathan Scheiman, Suhani Vora, Benjamin W Pruitt, Marcelle Tuttle, Eswar P R Iyer, Shuailiang Lin, Samira Kiani, Christopher D Guzman, Daniel J Wiegand, Dmitry Ter-Ovanesyan, Jonathan L Braff, Noah Davidsohn, Benjamin E Housden, Norbert Perrimon, Ron Weiss, John Aach, James J Collins, and George M Church. 2015. "Highly efficient Cas9-mediated transcriptional programming." Nat Methods. 2015 Apr; 12(4):326-8. PubMed ID: 25730490
Zachary L Sebo, Han B Lee, Ying Peng, and Yi Guo. 2013. "A simplified and efficient germline-specific CRISPR/Cas9 system for Drosophila genomic engineering." Fly. 2014;8(1):52-7. PubMed ID: 24141137
Fillip Port, Hui-Min Chen, Tzumin Lee, and Simon L. Bullock. 2014. "Optimixed CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila." PNAS. 2014 Jul 22;111(29):E2967-76. PubMed ID: 25002478
Shu Kondo, Ryu Ueda. 2013. "Highly Improved Gene Targeting by Germline-Specific Cas9 Expression in Drosophila." Genetics. 2013 Nov;195(3):715-21. PubMed ID: 24002648
Zhongsheng Yu, Mengda Ren, Zhanxiang Wang, Bo Zhang, Yikang S. Rong, Renjie Jiao, Guanjun Gao. 2013. "Highly efficient genome modifications mediated by CRISPR/Cas9 in Drosophila." Genetics. 2013 Sep;195(1):289-91. PubMed ID: 23833182
Scott J. Gratz, Alexander M. Cummings, Jennifer N. Nguyen, Danielle C. Hamm, Laura K. Donohue, Melissa M. Harrison, Jill Wildonger, Kate M. O’Connor-Giles. 2013. "Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease." Genetics. 2013 Aug;194(4):1029-35. Pubmed ID: 23709638
Andrew R. Bassett, Charlotte Tibbit, Chris P. Ponting, Ji-Long Liu. 2013. "Highly Efficient Targeted Mutagenesis of Drosophila with the CRISPR/Cas9 System." Cell Rep. 2013 Jul 11;4(1):220-8. PubMed ID: 23827738