CRISPR modified cell lines

CRISPR modified cell lines

We have begun to make CRISPR modified cell lines (such as knockouts) for the community. Please contact the Director if you are interested to learn about the possibility of custom cell line production. The following CRISPR modified cell lines made at the DRSC are available for distribution by the Drosophila Genome Resource Center (DGRC) in Bloomington, IN.

Knockout cell lines

S2R+-ZnT63C-KO, NHEJ-mediated knockout of ZnT63C. As described in PMID: 29223976. Ordering information: DGRC cell catalog #265.
S2R+-IA2-KO, NHEJ-meidated knockout of ia2. As described in PMID: 29223976. Ordering information: DGRC cell catalog #266.

GFP-tagged knock-in cell lines

We have an active project to make GFP-tagged cell lines, with an emphasis on visualization of various organelles and sub-cellular compartments. The following GFP knock-in cell lines made at the DRSC in collaboration with the Bellne lab are available for distribution by the DGRC in Bloomington, IN. 

Note that the parental cell line is positive for an mCherry fusion and Cas9, as the parental cell line is S2R+-MT::Cas9 (DGRC cell catalog #268), which is described in Viswanatha et al. 2018 (PubMed ID 30051818). This parental cell line was itself derived from DRSC cell line S2R+ NPT005 (DGRC cell catalog #229), which is described in Neumuller et al. 2012 (PubMed ID 22174071).

S2R+ with GFP::Cnx99a. Ordering information: DGRC cell catalog ID #273
S2R+ with GFP::Rab11. Ordering information: DGRC cell catalog ID #274
S2R+ with GFP::Polo. Ordering information: DGRC cell catalog ID #275
S2R+ with GFP::Gmap (clone #4). Ordering information: DGRC cell catalog ID #276
S2R+ with GFP::Gmap (clone $7). Ordering information: DGRC cell catalog ID #277
S2R+ with GFP::Fib (clone #11). Ordering information: DGRC cell catalog ID #278
S2R+ with GFP::Fib (clone #12). Ordering information: DGRC cell catalog ID #279
S2R+ with GFP::Golgin. Ordering information: DGRC cell catalog ID #280

These cell lines were made using constructs designed and provided by Kanca and Bellen. The cell lines were engineered, isolated, and validated at the DRSC. Validation testing included live-cell imaging, fixed-cell imaging (co-stained with an antibody, when possible), and molecular characterization of the insertion endpoints. Contact us for details.

Project funding from NIH ORIP R24 OD019847 "Next-generation Drosophila cell lines to elucidate the cellular basis of human diseases" (N. Perrimon, PI; A. Simcox, Co-PI).

Did you request these cells from the DGRC and use them in a study? If so, please acknowledge both the cell line developers and distribtors by citing NIH Grant 5R24OD019847, which supported production of the resource at DRSC/TRiP, and the Drosophila Genome Resource Center, NIH grant 2P40OD010949, as well as the relevant pulication (manuscript in preparation).


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.

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.

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.