CRISPR genome editing

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
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.
Huajin Wang, Michel Becuwe, Benjamin E Housden, Chandramohan Chitraju, Ashley J Porras, Morven M Graham, Xinran N Liu, Abdou Rachid Thiam, David B Savage, Anil K Agarwal, Abhimanyu Garg, Maria-Jesus Olarte, Qingqing Lin, Florian Fröhlich, Hans Kristian Hannibal-Bach, Srigokul Upadhyayula, Norbert Perrimon, Tomas Kirchhausen, Christer S Ejsing, Tobias C Walther, and Robert V Farese. 2016. “Seipin is required for converting nascent to mature lipid droplets.” Elife, 5.Abstract

How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation-the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs.

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.

Benjamin E Housden, Shuailiang Lin, and Norbert Perrimon. 2014. “Cas9-based genome editing in Drosophila.” Methods Enzymol, 546, Pp. 415-39.Abstract

Our ability to modify the Drosophila genome has recently been revolutionized by the development of the CRISPR system. The simplicity and high efficiency of this system allows its widespread use for many different applications, greatly increasing the range of genome modification experiments that can be performed. Here, we first discuss some general design principles for genome engineering experiments in Drosophila and then present detailed protocols for the production of CRISPR reagents and screening strategies to detect successful genome modification events in both tissue culture cells and animals.

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.

Xingjie Ren, Jin Sun, Benjamin E Housden, Yanhui Hu, Charles Roesel, Shuailiang Lin, Lu-Ping Liu, Zhihao Yang, Decai Mao, Lingzhu Sun, Qujie Wu, Jun-Yuan Ji, Jianzhong Xi, Stephanie E Mohr, Jiang Xu, Norbert Perrimon, and Jian-Quan Ni. 2013. “Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9.” Proc Natl Acad Sci U S A, 110, 47, Pp. 19012-7.Abstract

The ability to engineer genomes in a specific, systematic, and cost-effective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.

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.

Stephanie E Mohr, Jennifer A Smith, Caroline E Shamu, Ralph A Neumüller, and Norbert Perrimon. 2014. “RNAi screening comes of age: improved techniques and complementary approaches.” Nat Rev Mol Cell Biol, 15, 9, Pp. 591-600.Abstract

Gene silencing through sequence-specific targeting of mRNAs by RNAi has enabled genome-wide functional screens in cultured cells and in vivo in model organisms. These screens have resulted in the identification of new cellular pathways and potential drug targets. Considerable progress has been made to improve the quality of RNAi screen data through the development of new experimental and bioinformatics approaches. The recent availability of genome-editing strategies, such as the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system, when combined with RNAi, could lead to further improvements in screen data quality and follow-up experiments, thus promoting our understanding of gene function and gene regulatory networks.

Benjamin E Housden, Alexander J Valvezan, Colleen Kelley, Richelle Sopko, Yanhui Hu, Charles Roesel, Shuailiang Lin, Michael Buckner, Rong Tao, Bahar Yilmazel, Stephanie E Mohr, Brendan D Manning, and Norbert Perrimon. 2015. “Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi.” Sci Signal, 8, 393, Pp. rs9.Abstract

The tuberous sclerosis complex (TSC) family of tumor suppressors, TSC1 and TSC2, function together in an evolutionarily conserved protein complex that is a point of convergence for major cell signaling pathways that regulate mTOR complex 1 (mTORC1). Mutation or aberrant inhibition of the TSC complex is common in various human tumor syndromes and cancers. The discovery of novel therapeutic strategies to selectively target cells with functional loss of this complex is therefore of clinical relevance to patients with nonmalignant TSC and those with sporadic cancers. We developed a CRISPR-based method to generate homogeneous mutant Drosophila cell lines. By combining TSC1 or TSC2 mutant cell lines with RNAi screens against all kinases and phosphatases, we identified synthetic interactions with TSC1 and TSC2. Individual knockdown of three candidate genes (mRNA-cap, Pitslre, and CycT; orthologs of RNGTT, CDK11, and CCNT1 in humans) reduced the population growth rate of Drosophila cells lacking either TSC1 or TSC2 but not that of wild-type cells. Moreover, individual knockdown of these three genes had similar growth-inhibiting effects in mammalian TSC2-deficient cell lines, including human tumor-derived cells, illustrating the power of this cross-species screening strategy to identify potential drug targets.