CRISPRa

flySAM

Missed us at ADRC 2018? View our workshop slides!

April 19, 2018
Thank you to all those who attended our workshop at last week's Annual Drosophila Research Conference in Philadelphia, PA, USA. It was great to talk fly stocks, cell screens, and bioinformatics with the community. We are here to help and look forward to continued feedback on the resources we are building to empower your research. PDFs of our workshop presentations are attached to this news item. The slides will help you learn more about our in vivo resources for CRISPR, new pooled cell-based CRISPR screen technology, and bioinformatics resources at our facility.  Feel free to contact... Read more about Missed us at ADRC 2018? View our workshop slides!
2018 Apr 13

DRSC & TRiP Workshop at ADRC

1:45pm to 3:45pm

Location: 

Philadelphia, PA, USA
The DRSC & TRiP will be hosting a workshop at the Annual Drosophila Research Conference in Philadelphia, PA. The workshop is scheduled for Friday, April 13th from 1:45 to 3:45 PM. Come hear from DRSC & TRiP leaders Norbert Perrimon, Jonathan Zirin (organizer), Claire Yanhui Hu, and Stephanie Mohr. At the workshop, you will learn about new opportunities for community nomination and experiments using CRISPR knockout and activation, as well as learn what's new and popular among our online software and database tools. There will be something for everyone -- we will provide information... Read more about DRSC & TRiP Workshop at ADRC
2017 May 10

Presentation at Genome Editing and Trangenic Congress USA

10:00am

Location: 

Boston, MA

Stephanie will be presenting at the Genome Editing and Transgenic Congress USA in Boston, MA, on May 10, 2017 (time TBD). She plans to cover the following topics.

  • Strategy and results of optimization of Cas9 fusion proteins for CRISPR activation in cells and in vivo
  • Genome-scale design of single guide RNAs (sgRNAs) for CRISPR activation
  • Use of CRISPR activation in Drosophila for large-scale gene function discovery and disease modeling
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.

Shuailiang Lin, Ben Ewen-Campen, Xiaochun Ni, Benjamin E Housden, and Norbert Perrimon. 2015. “In Vivo Transcriptional Activation Using CRISPR/Cas9 in Drosophila.” Genetics, 201, 2, Pp. 433-42.Abstract

A number of approaches for Cas9-mediated transcriptional activation have recently been developed, allowing target genes to be overexpressed from their endogenous genomic loci. However, these approaches have thus far been limited to cell culture, and this technique has not been demonstrated in vivo in any animal. The technique involving the fewest separate components, and therefore the most amenable to in vivo applications, is the dCas9-VPR system, where a nuclease-dead Cas9 is fused to a highly active chimeric activator domain. In this study, we characterize the dCas9-VPR system in Drosophila cells and in vivo. We show that this system can be used in cell culture to upregulate a range of target genes, singly and in multiplex, and that a single guide RNA upstream of the transcription start site can activate high levels of target transcription. We observe marked heterogeneity in guide RNA efficacy for any given gene, and we confirm that transcription is inhibited by guide RNAs binding downstream of the transcription start site. To demonstrate one application of this technique in cells, we used dCas9-VPR to identify target genes for Twist and Snail, two highly conserved transcription factors that cooperate during Drosophila mesoderm development. In addition, we simultaneously activated both Twist and Snail to identify synergistic responses to this physiologically relevant combination. Finally, we show that dCas9-VPR can activate target genes and cause dominant phenotypes in vivo, providing the first demonstration of dCas9 activation in a multicellular animal. Transcriptional activation using dCas9-VPR thus offers a simple and broadly applicable technique for a variety of overexpression studies.