The Biological General Repository for Interaction Datasets (BioGRID: https://thebiogrid.org) is an open access database dedicated to the curation and archival storage of protein, genetic and chemical interactions for all major model organism species and humans. As of September 2018 (build 3.4.164), BioGRID contains records for 1 598 688 biological interactions manually annotated from 55 809 publications for 71 species, as classified by an updated set of controlled vocabularies for experimental detection methods. BioGRID also houses records for >700 000 post-translational modification sites. BioGRID now captures chemical interaction data, including chemical-protein interactions for human drug targets drawn from the DrugBank database and manually curated bioactive compounds reported in the literature. A new dedicated aspect of BioGRID annotates genome-wide CRISPR/Cas9-based screens that report gene-phenotype and gene-gene relationships. An extension of the BioGRID resource called the Open Repository for CRISPR Screens (ORCS) database (https://orcs.thebiogrid.org) currently contains over 500 genome-wide screens carried out in human or mouse cell lines. All data in BioGRID is made freely available without restriction, is directly downloadable in standard formats and can be readily incorporated into existing applications via our web service platforms. BioGRID data are also freely distributed through partner model organism databases and meta-databases.

The BioGRID interaction database: 2019 update

Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen, we further describe strategies for confirming the screening phenotype, as well as genetic perturbation, through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9-15 weeks, followed by 4-5 weeks of validation.

Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening

Several recent studies in a number of model systems including zebrafish, Arabidopsis, and mouse have revealed phenotypic differences between knockouts (i.e., mutants) and knockdowns (e.g., antisense-treated animals). These differences have been attributed to a number of reasons including off-target effects of the antisense reagents. An alternative explanation was recently proposed based on a zebrafish study reporting that genetic compensation was observed in egfl7 mutant but not knockdown animals. Dosage compensation was first reported in Drosophila in 1932, and genetic compensation in response to a gene knockout was first reported in yeast in 1969. Since then, genetic compensation has been documented many times in a number of model organisms; however, our understanding of the underlying molecular mechanisms remains limited. In this review, we revisit studies reporting genetic compensation in higher eukaryotes and outline possible molecular mechanisms, which may include both transcriptional and posttranscriptional processes.

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Genetic compensation: A phenomenon in search of mechanisms.

The BioGRID (Biological General Repository for Interaction Datasets, thebiogrid.org) is an open-access database resource that houses manually curated protein and genetic interactions from multiple species including yeast, worm, fly, mouse, and human. The ~1.93 million curated interactions in BioGRID can be used to build complex networks to facilitate biomedical discoveries, particularly as related to human health and disease. All BioGRID content is curated from primary experimental evidence in the biomedical literature, and includes both focused low-throughput studies and large high-throughput datasets. BioGRID also captures protein post-translational modifications and protein or gene interactions with bioactive small molecules including many known drugs. A built-in network visualization tool combines all annotations and allows users to generate network graphs of protein, genetic and chemical interactions. In addition to general curation across species, BioGRID undertakes themed curation projects in specific aspects of cellular regulation, for example the ubiquitin-proteasome system, as well as specific disease areas, such as for the SARS-CoV-2 virus that causes COVID-19 severe acute respiratory syndrome. A recent extension of BioGRID, named the Open Repository of CRISPR Screens (ORCS, orcs.thebiogrid.org), captures single mutant phenotypes and genetic interactions from published high throughput genome-wide CRISPR/Cas9-based genetic screens. BioGRID-ORCS contains datasets for over 1,042 CRISPR screens carried out to date in human, mouse and fly cell lines. The biomedical research community can freely access all BioGRID data through the web interface, standardized file downloads, or via model organism databases and partner meta-databases.

The BioGRID database: A comprehensive biomedical resource of curated protein, genetic, and chemical interactions.

The creation of genome-wide libraries for CRISPR knockout (CRISPRko), interference (CRISPRi), and activation (CRISPRa) has enabled the systematic interrogation of gene function. Here, we show that our recently-described CRISPRko library (Brunello) is more effective than previously published libraries at distinguishing essential and non-essential genes, providing approximately the same perturbation-level performance improvement over GeCKO libraries as GeCKO provided over RNAi. Additionally, we present genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese), and show in negative selection screens that Dolcetto, with fewer sgRNAs per gene, outperforms existing CRISPRi libraries and achieves comparable performance to CRISPRko in detecting essential genes. We also perform positive selection CRISPRa screens and demonstrate that Calabrese outperforms the SAM approach at identifying vemurafenib resistance genes. We further compare CRISPRa to genome-scale libraries of open reading frames (ORFs). Together, these libraries represent a suite of genome-wide tools to efficiently interrogate gene function with multiple modalities.

Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities

We compared the ability of short hairpin RNA (shRNA) and CRISPR/Cas9 screens to identify essential genes in the human chronic myelogenous leukemia cell line K562. We found that the precision of the two libraries in detecting essential genes was similar and that combining data from both screens improved performance. Notably, results from the two screens showed little correlation, which can be partially explained by the identification of distinct essential biological processes with each technology.

Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes

Broad-spectrum antiviral drugs targeting host processes could potentially treat a wide range of viruses while reducing the likelihood of emergent resistance. Despite great promise as therapeutics, such drugs remain largely elusive. Here we used parallel genome-wide high-coverage short hairpin RNA (shRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screens to identify the cellular target and mechanism of action of GSK983, a potent broad-spectrum antiviral with unexplained cytotoxicity. We found that GSK983 blocked cell proliferation and dengue virus replication by inhibiting the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). Guided by mechanistic insights from both genomic screens, we found that exogenous deoxycytidine markedly reduced GSK983 cytotoxicity but not antiviral activity, providing an attractive new approach to improve the therapeutic window of DHODH inhibitors against RNA viruses. Our results highlight the distinct advantages and limitations of each screening method for identifying drug targets, and demonstrate the utility of parallel knockdown and knockout screens for comprehensive probing of drug activity.

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Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification

Over 160 chiral vicinal bromochlorinated natural products have been identified; however, a lack of synthetic methods for the selective incorporation of halogens into organic molecules has hindered their synthesis. Here we disclose the first total synthesis and structural confirmation of isoplocamenone and plocamenone, as well as the first selective and scalable synthesis of the preclinical anticancer natural product halomon. The synthesis of these inter-halogenated compounds has been enabled by our recently developed chemo-, regio-, and enantioselective dihalogenation reaction.

Highly Selective Synthesis of Halomon, Plocamenone, and Isoplocamenone

Compounds and methods are provided for the treatment of pathogenic virus infections or cancer. The formulations combine an inhibitor of de novo pyrimidine synthesis, and an inhibitor of a pyrimidine salvage pathway enzyme.

Richard M Deans

Papers

Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes

Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification

Highly Selective Synthesis of Halomon, Plocamenone, and Isoplocamenone

Use of a dhodh inhibitor in combination with an inhibitor of pyrimidine salvage