CRISPR genome editing

2022
Justin A. Bosch and Norbert Perrimon. 2022. “Prime Editing for Precise Genome Engineering in Drosophila.” In Drosophila: Methods and Protocols, edited by Christian Dahmann, Pp. 113 - 134. New York, NY: Springer US. Publisher's VersionAbstract
Editing the Drosophila genome is incredibly useful for gene functional analysis. However, compared to gene knockouts, precise gene editing is difficult to achieve. Prime editing, a recently described CRISPR/Cas9-based technique, has the potential to make precise editing simpler and faster, and produce less errors than traditional methods. Initially described in mammalian cells, prime editing is functional in Drosophila somatic and germ cells. Here, we outline steps to design, generate, and express prime editing components in transgenic flies. Furthermore, we highlight a crossing scheme to produce edited fly stocks in less than 3 months.
Jonathan Zirin, Justin Bosch, Raghuvir Viswanatha, Stephanie E Mohr, and Norbert Perrimon. 2022. “State-of-the-art CRISPR for in vivo and cell-based studies in Drosophila.” Trends Genet, 38, 5, Pp. 437-453.Abstract
For more than 100 years, the fruit fly, Drosophila melanogaster, has served as a powerful model organism for biological and biomedical research due to its many genetic and physiological similarities to humans and the availability of sophisticated technologies used to manipulate its genome and genes. The Drosophila research community quickly adopted CRISPR technologies and, in the 8 years since the first clustered regularly interspaced short palindromic repeats (CRISPR) publications in flies, has explored and innovated methods for mutagenesis, precise genome engineering, and beyond. Moreover, the short lifespan and ease of genetics have made Drosophila an ideal testing ground for in vivo applications and refinements of the rapidly evolving set of CRISPR-associated (CRISPR-Cas) tools. Here, we review innovations in delivery of CRISPR reagents, increased efficiency of cutting and homology-directed repair (HDR), and alternatives to standard Cas9-based approaches. While the focus is primarily on in vivo systems, we also describe the role of Drosophila cultured cells as both an indispensable first step in the process of assessing new CRISPR technologies and a platform for genome-wide CRISPR pooled screens.
2021
Hans M. Dalton, Raghuvir Viswanatha, Ricky Brathwaite Jr., Jae Sophia Zuno, Stephanie E Mohr, Norbert Perrimon, and Clement Y. Chow. 12/4/2021. “A genome-wide CRISPR screen identifies the glycosylation enzyme DPM1 as a modifier of DPAGT1 deficiency and ER stress.” BioRxiv. Publisher's VersionAbstract
Partial loss-of-function mutations in glycosylation pathways underlie a set of rare diseases called Congenital Disorders of Glycosylation (CDGs). In particular, DPAGT1 CDG is caused by mutations in the gene encoding the first step in N-glycosylation, DPAGT1, and this disorder currently lacks effective therapies. To identify potential therapeutic targets for DPAGT1-CDG, we performed CRISPR knockout screens in Drosophila cells for genes associated with better survival and glycoprotein levels under DPAGT1 inhibition. We identified hundreds of candidate genes that may be of therapeutic benefit. Intriguingly, inhibition of the mannosyltransferase Dpm1, or its downstream glycosylation pathways, could rescue two in vivo models of DPAGT1 inhibition and ER stress, even though impairment of these pathways alone usually cause CDGs. While both in vivo models ostensibly cause ER stress (through DPAGT1 inhibition or a misfolded protein), we found a novel difference in fructose metabolism that may indicate glycolysis as a modulator of DPAGT1-CDG. Our results provide new therapeutic targets for DPAGT1-CDG, include the unique finding of Dpm1-related pathways rescuing DPAGT1 inhibition, and reveal a novel interaction between fructose metabolism and ER stress.
2021.12.03.471178v1.full_.pdf
Raghuvir Viswanatha, Enzo Mameli, Jonathan Rodiger, Pierre Merckaert, Fabiana Feitosa-Suntheimer, Tonya M Colpitts, Stephanie E Mohr, Yanhui Hu, and Norbert Perrimon. 11/24/2021. “Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos.” Nat Commun, 12, 1, Pp. 6825.Abstract
Mosquito-borne diseases present a worldwide public health burden. Current efforts to understand and counteract them have been aided by the use of cultured mosquito cells. Moreover, application in mammalian cells of forward genetic approaches such as CRISPR screens have identified essential genes and genes required for host-pathogen interactions, and in general, aided in functional annotation of genes. An equivalent approach for genetic screening of mosquito cell lines has been lacking. To develop such an approach, we design a new bioinformatic portal for sgRNA library design in several mosquito genomes, engineer mosquito cell lines to express Cas9 and accept sgRNA at scale, and identify optimal promoters for sgRNA expression in several mosquito species. We then optimize a recombination-mediated cassette exchange system to deliver CRISPR sgRNA and perform pooled CRISPR screens in an Anopheles cell line. Altogether, we provide a platform for high-throughput genome-scale screening in cell lines from disease vector species.
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Raghuvir Viswanatha, Enzo Mameli, Jonathan Rodiger, Pierre Merckaert, Fabiana Feitosa-Suntheimer, Tonya M. Colpitts, Stephanie E. Mohr, Yanhui Hu, and Norbert Perrimon. 3/30/2021. “Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos [NOTE: A modified final version was published in Nat Comm and is now available.].” bioRxiv. Publisher's VersionAbstract
Mosquito-borne diseases present a worldwide public health burden. Genome-scale screening tools that could inform our understanding of mosquitos and their control are lacking. Here, we adapt a recombination-mediated cassette exchange system for delivery of CRISPR sgRNA libraries into cell lines from several mosquito species and perform pooled CRISPR screens in an Anopheles cell line. To implement this method, we engineered modified mosquito cell lines, validated promoters and developed bioinformatics tools for multiple mosquito species.Competing Interest StatementThe authors have declared no competing interest.
2021.03.29.437496v2.full_.pdf
Xuechun Feng, Víctor López Del Amo, Enzo Mameli, Megan Lee, Alena L Bishop, Norbert Perrimon, and Valentino M Gantz. 2021. “Optimized CRISPR tools and site-directed transgenesis towards gene drive development in Culex quinquefasciatus mosquitoes.” Nat Commun, 12, 1, Pp. 2960.Abstract
Culex mosquitoes are a global vector for multiple human and animal diseases, including West Nile virus, lymphatic filariasis, and avian malaria, posing a constant threat to public health, livestock, companion animals, and endangered birds. While rising insecticide resistance has threatened the control of Culex mosquitoes, advances in CRISPR genome-editing tools have fostered the development of alternative genetic strategies such as gene drive systems to fight disease vectors. However, though gene-drive technology has quickly progressed in other mosquitoes, advances have been lacking in Culex. Here, we develop a Culex-specific Cas9/gRNA expression toolkit and use site-directed homology-based transgenesis to generate and validate a Culex quinquefasciatus Cas9-expressing line. We show that gRNA scaffold variants improve transgenesis efficiency in both Culex quinquefasciatus and Drosophila melanogaster and boost gene-drive performance in the fruit fly. These findings support future technology development to control Culex mosquitoes and provide valuable insight for improving these tools in other species.
s41467-021-23239-0.pdf
J. A. Bosch, G. Birchak, and N. Perrimon. 2021. “Precise genome engineering in Drosophila using prime editing.” Proc Natl Acad Sci U S A, 118.Abstract
Precise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double-strand breaks or donor DNA and has low off-target effects. Here, we applied prime editing for the model organism Drosophila melanogaster and developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, we precisely introduce premature stop codons in three classical visible marker genes, ebony, white, and forked Furthermore, by restricting editing to germ cells, we demonstrate efficient germ-line transmission of a precise edit in ebony to 36% of progeny. Our results suggest that prime editing is a useful system in Drosophila to study gene function, such as engineering precise point mutations, deletions, or epitope tags.
2020
R. Viswanatha, M. Zaffagni, J. Zirin, N. Perrimon, and S. Kadener. 11/1/2020. “CRISPR-Cas13 mediated Knock Down in Drosophila cultured cells.” BioRxiv.Abstract
Manipulation of gene expression is one of the best approaches for studying gene function in vivo. CRISPR-Cas13 has the potential to be a powerful technique for manipulating RNA expression in diverse animal systems in vivo, including Drosophila melanogaster. Studies using Cas13 in mammalian cell lines for gene knockdown showed increased on-target efficiency and decreased off-targeting relative to RNAi. Moreover, catalytically inactive Cas13 fusions can be used to image RNA molecules, install precise changes to the epitranscriptome, or alter splicing. However, recent studies have suggested that there may be limitations to the deployment of these tools in Drosophila, so further optimization of the system is required. Here, we report a new set of PspCas13b and RfxCas13d expression constructs and use these reagents to successfully knockdown both reporter and endogenous transcripts in Drosophila cells. As toxicity issues have been reported with high level of Cas13, we effectively decreased PspCas13b expression without impairing its function by tuning down translation. Furthermore, we altered the spatial activity of both PspCas13b and RfxCas13d by introducing Nuclear Exportation Sequences (NES) and Nuclear Localization Sequences (NLS) while maintaining activity. Finally, we generated a stable cell line expressing RfxCas13d under the inducible metallothionein promoter, establishing a useful tool for high-throughput genetic screening. Thus, we report new reagents for performing RNA CRISPR-Cas13 experiments in Drosophila, providing additional Cas13 expression constructs that retain activity.
2020.11.01.364166v1.full_.pdf
Yanhui Hu, Aram Comjean, Jonathan Rodiger, Yifang Liu, Yue Gao, Verena Chung, Jonathan Zirin, Norbert Perrimon, and Stephanie E Mohr. 2020. “FlyRNAi.org-the database of the Drosophila RNAi screening center and transgenic RNAi project: 2021 update.” Nucleic Acids Res.Abstract
The FlyRNAi database at the Drosophila RNAi Screening Center and Transgenic RNAi Project (DRSC/TRiP) provides a suite of online resources that facilitate functional genomics studies with a special emphasis on Drosophila melanogaster. Currently, the database provides: gene-centric resources that facilitate ortholog mapping and mining of information about orthologs in common genetic model species; reagent-centric resources that help researchers identify RNAi and CRISPR sgRNA reagents or designs; and data-centric resources that facilitate visualization and mining of transcriptomics data, protein modification data, protein interactions, and more. Here, we discuss updated and new features that help biological and biomedical researchers efficiently identify, visualize, analyze, and integrate information and data for Drosophila and other species. Together, these resources facilitate multiple steps in functional genomics workflows, from building gene and reagent lists to management, analysis, and integration of data.
gkaa936.pdf
Jonathan Zirin, Yanhui Hu, Luping Liu, Donghui Yang-Zhou, Ryan Colbeth, Dong Yan, Ben Ewen-Campen, Rong Tao, Eric Vogt, Sara VanNest, Cooper Cavers, Christians Villalta, Aram Comjean, Jin Sun, Xia Wang, Yu Jia, Ruibao Zhu, Ping Peng, Jinchao Yu, Da Shen, Yuhao Qiu, Limmond Ayisi, Henna Ragoowansi, Ethan Fenton, Senait Efrem, Annette Parks, Kuniaki Saito, Shu Kondo, Liz Perkins, Stephanie E Mohr, Jianquan Ni, and Norbert Perrimon. 2020. “Large-Scale Transgenic Resource Collections for Loss- and Gain-of-Function Studies.” Genetics.Abstract
The Transgenic RNAi Project (TRiP), a functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNAi fly stocks. To date, the TRiP has generated >15,000 RNAi fly stocks. As this covers most genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express sgRNAs targeting upstream of a gene transcription start site. Gene activation is triggered by co-expression of catalytically dead Cas9 (dCas9) fused to an activator domain, either VP64-p65-Rta (VPR) or Synergistic Activation Mediator (SAM). TRiP-KO stocks express one or two sgRNAs targeting the coding sequence of a gene or genes. Cutting is triggered by co-expression of Cas9, allowing for generation of indels in both germline and somatic tissue. To date, we have generated more than 5,000 CRISPR-OE or -KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.
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.
2019
Justin A. Bosch, Ryan Colbeth, Jonathan Zirin, and Norbert Perrimon. 5/16/2019. “Gene knock-ins in Drosophila using homology-independent insertion of universal donor plasmids.” BioRxiv. Publisher's Version 2019_BioRxiv_Bosch .pdf
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.
elife-51539-v2.pdf
Hilary E Nicholson, Zeshan Tariq, Benjamin E Housden, Rebecca B Jennings, Laura A Stransky, Norbert Perrimon, Sabina Signoretti, and William G Kaelin. 2019. “HIF-independent synthetic lethality between CDK4/6 inhibition and VHL loss across species.” Sci Signal, 12, 601.Abstract
Inactivation of the tumor suppressor gene is the signature initiating event in clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, and causes the accumulation of hypoxia-inducible factor 2α (HIF-2α). HIF-2α inhibitors are effective in some ccRCC cases, but both de novo and acquired resistance have been observed in the laboratory and in the clinic. Here, we identified synthetic lethality between decreased activity of cyclin-dependent kinases 4 and 6 (CDK4/6) and inactivation in two species (human and ) and across diverse human ccRCC cell lines in culture and xenografts. Although HIF-2α transcriptionally induced the CDK4/6 partner cyclin D1, HIF-2α was not required for the increased CDK4/6 requirement of ccRCC cells. Accordingly, the antiproliferative effects of CDK4/6 inhibition were synergistic with HIF-2α inhibition in HIF-2α-dependent ccRCC cells and not antagonistic with HIF-2α inhibition in HIF-2α-independent cells. These findings support testing CDK4/6 inhibitors as treatments for ccRCC, alone and in combination with HIF-2α inhibitors.
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.
Chiao-Lin Chen, Jonathan Rodiger, Verena Chung, Raghuvir Viswanatha, Stephanie E Mohr, Yanhui Hu, and Norbert Perrimon. 2019. “SNP-CRISPR: A Web Tool for SNP-Specific Genome Editing.” G3 (Bethesda).Abstract
CRISPR-Cas9 is a powerful genome editing technology in which a short guide RNA (sgRNA) confers target site specificity to achieve Cas9-mediated genome editing. Numerous sgRNA design tools have been developed based on reference genomes for humans and model organisms. However, existing resources are not optimal as genetic mutations or single nucleotide polymorphisms (SNPs) within the targeting region affect the efficiency of CRISPR-based approaches by interfering with guide-target complementarity. To facilitate identification of sgRNAs (1) in non-reference genomes, (2) across varying genetic backgrounds, or (3) for specific targeting of SNP-containing alleles, for example, disease relevant mutations, we developed a web tool, SNP-CRISPR (https://www.flyrnai.org/tools/snp_crispr/). SNP-CRISPR can be used to design sgRNAs based on public variant data sets or user-identified variants. In addition, the tool computes efficiency and specificity scores for sgRNA designs targeting both the variant and the reference. Moreover, SNP-CRISPR provides the option to upload multiple SNPs and target single or multiple nearby base changes simultaneously with a single sgRNA design. Given these capabilities, SNP-CRISPR has a wide range of potential research applications in model systems and potential applications for design of sgRNAs for disease-associated mutant correction.
2018
Naoki Okamoto, Raghuvir Viswanatha, Riyan Bittar, Zhongchi Li, Sachiko Haga-Yamanaka, Norbert Perrimon, and Naoki Yamanaka. 2018. “A Membrane Transporter Is Required for Steroid Hormone Uptake in Drosophila.” Dev Cell, 47, 3, Pp. 294-305.e7.Abstract
Steroid hormones are a group of lipophilic hormones that are believed to enter cells by simple diffusion to regulate diverse physiological processes through intracellular nuclear receptors. Here, we challenge this model in Drosophila by demonstrating that Ecdysone Importer (EcI), a membrane transporter identified from two independent genetic screens, is involved in cellular uptake of the steroid hormone ecdysone. EcI encodes an organic anion transporting polypeptide of the evolutionarily conserved solute carrier organic anion superfamily. In vivo, EcI loss of function causes phenotypes indistinguishable from ecdysone- or ecdysone receptor (EcR)-deficient animals, and EcI knockdown inhibits cellular uptake of ecdysone. Furthermore, EcI regulates ecdysone signaling in a cell-autonomous manner and is both necessary and sufficient for inducing ecdysone-dependent gene expression in culture cells expressing EcR. Altogether, our results challenge the simple diffusion model for cellular uptake of ecdysone and may have wide implications for basic and medical aspects of steroid hormone studies.
2017
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.
2016
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.

2016_Nat Rev Gene_Housden.pdf
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.

2016_FEBS_Mohr.pdf

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