Cell-based CRISPR

Baolong Xia, Gabriel Amador, Raghuvir Viswanatha, Jonathan Zirin, Stephanie E Mohr, and Norbert Perrimon. 2020. “CRISPR-based engineering of gene knockout cells by homology-directed insertion in polyploid Drosophila S2R+ cells.” Nat Protoc, 15, 10, Pp. 3478-3498.Abstract
Precise and efficient genome modifications provide powerful tools for biological studies. Previous CRISPR gene knockout methods in cell lines have relied on frameshifts caused by stochastic insertion/deletion in all alleles. However, this method is inefficient for genes with high copy number due to polyploidy or gene amplification because frameshifts in all alleles can be difficult to generate and detect. Here we describe a homology-directed insertion method to knockout genes in the polyploid Drosophila S2R+ cell line. This protocol allows generation of homozygous mutant cell lines using an insertion cassette which autocatalytically generates insertion mutations in all alleles. Knockout cells generated using this method can be directly identified by PCR without a need for DNA sequencing. This protocol takes 2-3 months and can be applied to other polyploid cell lines or high-copy-number genes.
Justin A Bosch, Shannon Knight, Oguz Kanca, Jonathan Zirin, Donghui Yang-Zhou, Yanhui Hu, Jonathan Rodiger, Gabriel Amador, Hugo J Bellen, Norbert Perrimon, and Stephanie E Mohr. 2020. “Use of the CRISPR-Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes.” Curr Protoc Mol Biol, 130, 1, Pp. e112.Abstract
The CRISPR-Cas9 system makes it possible to cause double-strand breaks in specific regions, inducing repair. In the presence of a donor construct, repair can involve insertion or 'knock-in' of an exogenous cassette. One common application of knock-in technology is to generate cell lines expressing fluorescently tagged endogenous proteins. The standard approach relies on production of a donor plasmid with ∼500 to 1000 bp of homology on either side of an insertion cassette that contains the fluorescent protein open reading frame (ORF). We present two alternative methods for knock-in of fluorescent protein ORFs into Cas9-expressing Drosophila S2R+ cultured cells, the single-stranded DNA (ssDNA) Drop-In method and the CRISPaint universal donor method. Both methods eliminate the need to clone a large plasmid donor for each target. We discuss the advantages and limitations of the standard, ssDNA Drop-In, and CRISPaint methods for fluorescent protein tagging in Drosophila cultured cells. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Knock-in into Cas9-positive S2R+ cells using the ssDNA Drop-In approach Basic Protocol 2: Knock-in into Cas9-positive S2R+ cells by homology-independent insertion of universal donor plasmids that provide mNeonGreen (CRISPaint method) Support Protocol 1: sgRNA design and cloning Support Protocol 2: ssDNA donor synthesis Support Protocol 3: Transfection using Effectene Support Protocol 4: Electroporation of S2R+-MT::Cas9 Drosophila cells Support Protocol 5: Single-cell isolation of fluorescent cells using FACS.
Raghuvir Viswanatha, Roderick Brathwaite, Yanhui Hu, Zhongchi Li, Jonathan Rodiger, Pierre Merckaert, Verena Chung, Stephanie E Mohr, and Norbert Perrimon. 2019. “Pooled CRISPR Screens in Drosophila Cells.” Curr Protoc Mol Biol, 129, 1, Pp. e111.Abstract
High-throughput screens in Drosophila melanogaster cell lines have led to discovery of conserved gene functions related to signal transduction, host-pathogen interactions, ion transport, and more. CRISPR/Cas9 technology has opened the door to new types of large-scale cell-based screens. Whereas array-format screens require liquid handling automation and assay miniaturization, pooled-format screens, in which reagents are introduced at random and in bulk, can be done in a standard lab setting. We provide a detailed protocol for conducting and evaluating genome-wide CRISPR single guide RNA (sgRNA) pooled screens in Drosophila S2R+ cultured cells. Specifically, we provide step-by-step instructions for library design and production, optimization of cytotoxin-based selection assays, genome-scale screening, and data analysis. This type of project takes ∼3 months to complete. Results can be used in follow-up studies performed in vivo in Drosophila, mammalian cells, and/or other systems. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Pooled-format screening with Cas9-expressing Drosophila S2R+ cells in the presence of cytotoxin Support Protocol 1: Optimization of cytotoxin concentration for Drosophila cell screening Support Protocol 2: CRISPR sgRNA library design and production for Drosophila cell screening Support Protocol 3: Barcode deconvolution and analysis of screening data.
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.
Graphical image of tissue culture, fly pushing, and computer, and the team of people who work with them

DRSC-Biomedical Technology Research Resource

October 21, 2019

We are pleased to announce that we have been funded by NIH NIGMS to form the Drosophila Research & Screening Center-Biomedical Technology Research Resource (DRSC-BTRR). The P41-funded DRSC-BTRR (N. Perrimon, PI; S. Mohr, Co-I) builds upon and extends past goals of the Drosophila RNAi Screening Center.

As the DRSC-BTRR, we are working together with collaborators whose 'driving biomedical projects' inform development of new technologies at the DRSC. At the same time, we continue to support Drosophila cell-based RNAi and CRIPSR knockout screens and related...

Read more about DRSC-Biomedical Technology Research Resource
Photo of 384-well assay plates

Drosophila cell screen with DRSC reagent library contributes to identification of new therapeutic target for renal cancer

October 7, 2019

We here at the DRSC/TRiP are thrilled to see this study from Hilary Nicholson et al. published in Science Signaling.

The study provides a great example of how screens in Drosophila cultured cells can be used as part of a cross-species platform aimed at discovery of new targets for disease treatment. The work represents a collaboration between the laboratory of 2019 Nobel Prize winner W. Kaelin and DRSC PI N. Perrimon.

...

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2018 Dec 08

DRSC&TRiP at ASCB|EMBO 2018

Sat Dec 8 (All day) to Wed Dec 12 (All day)

Location: 

San Diego, CA, USA
The DRSC & TRiP will be represented at the ASCB|EMBO 2018 conference in the form of a poster that includes information about our collaboration with O. Kanca in the H. Bellen lab at Baylor College of Medicine to tag Drosophila S2R+ cell lines with GFP using a CRISPR-based strategy. We have 'painted' a number of subcellular localizations green with this approach! If you're at the meeting, check out P1482 at Board Number B494, presented by Baolong Xia of the Perrimon lab.
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!
Cartoon of essential gene pooled screen (made using BioRender.io)

Pooled-format CRISPR screens in Drosophila cells

March 22, 2018

The DRSC/TRiP-FGR is pleased to support collaborations on pooled CRISPR screens using the method recently, reported in eLife by Viswanatha et al. (PDF download file below).

From the abstract: "... Here, we developed a site-specific integration strategy for library delivery and performed a genome-wide CRISPR knockout screen in Drosophila S2R+ cells. Under basal growth conditions, 1235 genes were essential for cell fitness...

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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

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