in vivo CRISPR (TRiP)

Quick link to Find CRISPR sgRNA search
Quick link to TRiP-CRISPR sgRNA stock database

Using the TRiP pipeline, we are actively developing CRISPR fly stocks and resources for gene editing and other applications. These resources facilitate knockout, up-regulation (CRIPSRa), and other Drosophila in vivo CRISPR applications.

TRiP-CRISPR fly stock collections

Function

sgRNA Vectors

Cross to

Notes

TRiP-OE

VPR

Gene Activation

pCFD4

Gal4+dCas9-VPR

See TRiP-CRISPR Toolbox

flySAM1.0

Gene Activation

U6B-sgRNA2.0

Gal4+flySAM

See TRiP-CRISPR Toolbox

flySAM2.0

Gene Activation

flySAM2.0

Gal4

flySAM2.0 lines contain both sgRNA and dCas9 (flySAM)

TRiP-KO

Gene Cutting

pCFD3, pCFD4

Gal4+Cas9

See TRiP-CRISPR Toolbox

Search the DRSC/TRiP sgRNA Stock Database for TRiP-OE and TRiP-KO stocks
Nominate genes for production for TRiP-OE and TRiP-KO stocks
Download a .XLS file of currently available TRiP-OE and TRiP-KO stocks
Protocols for cloning, sequencing, and using TRiP-CRISPR lines
Online tools for gRNA design and efficiency prediction

See links below to relevant online tools, reagents, protocols, publications, and more.

in vivo CRISPR

Protocols

Contact Us

Please contact us for any questions.

Publications

Jun Xu, Ah-Ram Kim, Ross W. Cheloha, Fabian A. Fischer, Joshua Shing Shun Li, Yuan Feng, Emily Stoneburner, Richard Binari, Stephanie E. Mohr, Jonathan Zirin, Hidde Ploegh, and Norbert Perrimon. Working Paper. “Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies.” bioRxiv.Abstract
Expansion of the available repertoire of reagents for visualization and manipulation of proteins will help understand their function. Short epitope tags installed on proteins of interest and recognized by existing binders such as nanobodies facilitate protein studies by obviating the need to isolate new antibodies directed against them. Nanobodies have several advantages over conventional antibodies, as they can be expressed and used as tools for visualization and manipulation of proteins in vivo. Here, we combine the advantages of short epitopes (NanoTags) and nanobodies specific for them by characterizing two short (<15 aa) tags, 127D01 and VHH05, which are high-affinity targets of nanobodies. We demonstrate that these NanoTags and the nanobodies that recognize them can be used in Drosophila for in vivo protein detection and re-localization, direct and indirect immunofluorescence, immunoblotting, and immunoprecipitation. We further show that CRISPR-mediated gene targeting provides a straightforward approach to tagging endogenous proteins with the NanoTags. Single copies of the NanoTags, regardless of their location, suffice for detection. This versatile and validated toolbox of tags and nanobodies will serve as a resource for a wide array of applications, including functional studies in Drosophila and beyond.Competing Interest StatementThe authors have declared no competing interest.
R. Viswanatha, M. Zaffagni, J. Zirin, N. Perrimon, and S. Kadener. Working Paper. “CRISPR-Cas13 mediated Knock Down in Drosophila cultured cells.” BioRxiv.Abstract
Manipulation of gene expression is one of the best approaches for studying gene function in vivo. CRISPR-Cas13 has the potential to be a powerful technique for manipulating RNA expression in diverse animal systems in vivo, including Drosophila melanogaster. Studies using Cas13 in mammalian cell lines for gene knockdown showed increased on-target efficiency and decreased off-targeting relative to RNAi. Moreover, catalytically inactive Cas13 fusions can be used to image RNA molecules, install precise changes to the epitranscriptome, or alter splicing. However, recent studies have suggested that there may be limitations to the deployment of these tools in Drosophila, so further optimization of the system is required. Here, we report a new set of PspCas13b and RfxCas13d expression constructs and use these reagents to successfully knockdown both reporter and endogenous transcripts in Drosophila cells. As toxicity issues have been reported with high level of Cas13, we effectively decreased PspCas13b expression without impairing its function by tuning down translation. Furthermore, we altered the spatial activity of both PspCas13b and RfxCas13d by introducing Nuclear Exportation Sequences (NES) and Nuclear Localization Sequences (NLS) while maintaining activity. Finally, we generated a stable cell line expressing RfxCas13d under the inducible metallothionein promoter, establishing a useful tool for high-throughput genetic screening. Thus, we report new reagents for performing RNA CRISPR-Cas13 experiments in Drosophila, providing additional Cas13 expression constructs that retain activity.
J. A. Bosch, G. Birchak, and N. Perrimon. 2021. “Precise genome engineering in Drosophila using prime editing.” Proc Natl Acad Sci U S A, 118.Abstract
Precise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double-strand breaks or donor DNA and has low off-target effects. Here, we applied prime editing for the model organism Drosophila melanogaster and developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, we precisely introduce premature stop codons in three classical visible marker genes, ebony, white, and forked Furthermore, by restricting editing to germ cells, we demonstrate efficient germ-line transmission of a precise edit in ebony to 36% of progeny. Our results suggest that prime editing is a useful system in Drosophila to study gene function, such as engineering precise point mutations, deletions, or epitope tags.
Jonathan Zirin, Yanhui Hu, Luping Liu, Donghui Yang-Zhou, Ryan Colbeth, Dong Yan, Ben Ewen-Campen, Rong Tao, Eric Vogt, Sara VanNest, Cooper Cavers, Christians Villalta, Aram Comjean, Jin Sun, Xia Wang, Yu Jia, Ruibao Zhu, Ping Peng, Jinchao Yu, Da Shen, Yuhao Qiu, Limmond Ayisi, Henna Ragoowansi, Ethan Fenton, Senait Efrem, Annette Parks, Kuniaki Saito, Shu Kondo, Liz Perkins, Stephanie E Mohr, Jianquan Ni, and Norbert Perrimon. 2020. “Large-Scale Transgenic Resource Collections for Loss- and Gain-of-Function Studies.” Genetics.Abstract
The Transgenic RNAi Project (TRiP), a 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 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. Cutting is triggered by co-expression of Cas9, 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.
Oguz Kanca, Jonathan Zirin, Jorge Garcia-Marques, Shannon Marie Knight, Donghui Yang-Zhou, Gabriel Amador, Hyunglok Chung, Zhongyuan Zuo, Liwen Ma, Yuchun He, Wen-Wen Lin, Ying Fang, Ming Ge, Shinya Yamamoto, Karen L Schulze, Yanhui Hu, Allan C Spradling, Stephanie E Mohr, Norbert Perrimon, and Hugo J Bellen. 2019. “An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms.” Elife, 8.Abstract
We previously reported a CRISPR-mediated knock-in strategy into introns of genes, generating an - transgenic library for multiple uses (Lee et al., 2018b). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely . The approach is fast, cheap, and scalable.
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