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CRISPR modified cell lines

CRISPR modified cell lines

We are using CRISPR gene editing technologies to generate new cell lines as part of the funded project NIH ORIP R24 OD019847 "Next-generation Drosophila cell lines to elucidate the cellular basis of human diseases" (N. Perrimon, PI; A. Simcox, Co-PI).

GFP-tagged knock-in cell lines

As part of the ORIP-funded project, we are making 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
S2R+ with GFP::Arl8. Ordering information: DGRC cell catalog ID #291
S2R+ with GFP::Lam. Ordering information: DGRC cell catalog ID #292
S2R+ with GFP::Spin. Ordering information: DGRC cell catalog ID #293
S2R+ with GFP::Sec23. Ordering information: DGRC cell catalog ID #294
S2R+ with GFP::Tom20. Ordering information: DGRC cell catalog ID #302

These cell lines were made using constructs designed and provided by Kanca and Bellen (Baylor College of Medicine). 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.

In addition, C-terminal GFP knock-in cell lines were generated as described in a BioRxiv preprint from Bosch et al. (2019) using an 'armless' donor approach. Act5c::GFP, Tub84B::GFP, His2Av::GFP, and Lamin::GFP fusion cell lines were made using this approach and are being shared with the DGRC for distribution to the community.

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-mediated knockout of ia2. As described in PMID: 29223976. Ordering information: DGRC cell catalog #266.
S2R+-Apc-KO, two independent cell lines made with one method, DGRC cell catalog #271 and DGRC cell catalog #272
S2R+-Apc-KO, one additional cell line made with a different method, DGRC cell catalog #270
S2R+-gig-KO, DGRC cell catalog #297
S2R+-hairy-KO, DGRC cell catalog #298
S2R+-Tnks-KO, three independent cell lines, DGRC cell catalog #299, DGRC cell catalog #300, and DGRC cell catalog #301
S2R+-Tnks-KO, one additional cell line made with a different method, DGRC cell catalog #304

These cell lines have been sequence verified as containing only knockout alleles by PCR amplification of the target region followed by next-generation sequencing of the PCR amplicon, contig assembly, and comparison with the wild-type reference sequence (or, for KO alleles generated via knock-in, verified using PCR validation of the insertion allele). We wanted to make sure that the distribution copies of the cell lines are correct. To do this, for a subset of these cell lines, (1) the DGRC prepared genomic DNA from their distribution copies of the cell lines, (2) they shipped that gDNA to the DRSC/TRiP, and (3) we used that gDNA as template for PCR and NGS, and validated that all alleles are predicted to be gene knockout alleles.

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).

Publications

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
elife-51539-v2.pdf
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.
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