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

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.
Ben Ewen-Campen, Stephanie E Mohr, Yanhui Hu, and Norbert Perrimon. 10/9/2017. “Accessing the Phenotype Gap: Enabling Systematic Investigation of Paralog Functional Complexity with CRISPR.” Dev Cell, 43, 1, Pp. 6-9.Abstract
Single-gene knockout experiments can fail to reveal function in the context of redundancy, which is frequently observed among duplicated genes (paralogs) with overlapping functions. We discuss the complexity associated with studying paralogs and outline how recent advances in CRISPR will help address the "phenotype gap" and impact biomedical research.
Benjamin E Housden, Matthias Muhar, Matthew Gemberling, Charles A Gersbach, Didier YR Stainier, Geraldine Seydoux, Stephanie E Mohr, Johannes Zuber, and Norbert Perrimon. 10/31/2016. “Loss-of-function genetic tools for animal models: cross-species and cross-platform differences.” Nat Rev Genet. Publisher's VersionAbstract

Our understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses. LOF methods target DNA, RNA or protein to reduce or to ablate gene function. By analysing the phenotypes that are caused by these perturbations the wild-type function of genes can be elucidated. Although all LOF methods reduce gene activity, the choice of approach (for example, mutagenesis, CRISPR-based gene editing, RNA interference, morpholinos or pharmacological inhibition) can have a major effect on phenotypic outcomes. Interpretation of the LOF phenotype must take into account the biological process that is targeted by each method. The practicality and efficiency of LOF methods also vary considerably between model systems. We describe parameters for choosing the optimal combination of method and system, and for interpreting phenotypes within the constraints of each method.

Stephanie E Mohr, Yanhui Hu, Benjamin Ewen-Campen, Benjamin E Housden, Raghuvir Viswanatha, and Norbert Perrimon. 2016. “CRISPR guide RNA design for research applications.” FEBS J.Abstract

The rapid rise of CRISPR as a technology for genome engineering and related research applications has created a need for algorithms and associated online tools that facilitate design of on-target and effective guide RNAs (gRNAs). Here, we review the state-of-the-art in CRISPR gRNA design for research applications of the CRISPR-Cas9 system, including knockout, activation and inhibition. Notably, achieving good gRNA design is not solely dependent on innovations in CRISPR technology. Good design and design tools also rely on availability of high-quality genome sequence and gene annotations, as well as on availability of accumulated data regarding off-targets and effectiveness metrics. This article is protected by copyright. All rights reserved.

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