A public genome-scale lentiviral expression library of human ORFs
TL;DR: This work describes the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames encoded in a versatile Gateway vector system, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.
Abstract: Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments. We describe the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Using this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.
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TL;DR: The map uncovers significant interconnectivity between known and candidate cancer gene products, providing unbiased evidence for an expanded functional cancer landscape, while demonstrating how high-quality interactome models will help "connect the dots" of the genomic revolution.
1,220 citations
Cites background from "A public genome-scale lentiviral ex..."
...…advantage of the emergence of reference proteome maps (Kim et al., 2014; Wilhelm et al., 2014), a combination of nearly complete clone collections (Yang et al., 2011), rapid improvements in Y2H assay sensitivity, and emerging interaction-mapping technologies that drastically reduce cost (Caufield…...
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..., 2014), a combination of nearly complete clone collections (Yang et al., 2011), rapid improvements in Y2H assay sensitivity and emerging interaction-mapping technologies that drastically reduce cost (Caufield et al....
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TL;DR: Using high-throughput affinity-purification mass spectrometry, BioPlex is used to identify interacting partners for 2,594 human proteins in HEK293T cells and reveals associations among thousands of protein domains, suggesting a basis for examining structurally related proteins.
1,189 citations
Cites methods from "A public genome-scale lentiviral ex..."
...…in Figure 1A and described in the Supplemental Experimental Procedures, we have initiated high-throughput lentiviral expression and AP-MS profiling of C-terminally FLAG-HA-tagged baits from 13,000 protein-coding ORFs within the sequence-validated Human ORFEOME collection v. 8.1 (Yang et al., 2011)....
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...Protein Expression and Purification The sequence-validated Human ORFEOME v. 8.1 (Yang et al., 2011) was used to construct a lentiviral library containing 13,000 ORFs bearing C-terminal FLAG-HA epitopes....
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...1 (Yang et al., 2011) was used to construct a lentiviral library containing 13,000 ORFs bearing C-terminal FLAG-HA epitopes....
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TL;DR: An improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9 is described and demonstrated in activating endogenous coding and noncoding genes and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).
Abstract: The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. We demonstrate its utility in activating endogenous coding and noncoding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).
1,147 citations
TL;DR: With more than 56,000 candidate interactions, BioPlex 2.0 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization.
Abstract: The physiology of a cell can be viewed as the product of thousands of proteins acting in concert to shape the cellular response Coordination is achieved in part through networks of protein-protein interactions that assemble functionally related proteins into complexes, organelles, and signal transduction pathways Understanding the architecture of the human proteome has the potential to inform cellular, structural, and evolutionary mechanisms and is critical to elucidating how genome variation contributes to disease Here we present BioPlex 20 (Biophysical Interactions of ORFeome-derived complexes), which uses robust affinity purification-mass spectrometry methodology to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein-coding genes from the human genome, and constitutes, to our knowledge, the largest such network so far With more than 56,000 candidate interactions, BioPlex 20 contains more than 29,000 previously unknown co-associations and provides functional insights into hundreds of poorly characterized proteins while enhancing network-based analyses of domain associations, subcellular localization, and co-complex formation Unsupervised Markov clustering of interacting proteins identified more than 1,300 protein communities representing diverse cellular activities Genes essential for cell fitness are enriched within 53 communities representing central cellular functions Moreover, we identified 442 communities associated with more than 2,000 disease annotations, placing numerous candidate disease genes into a cellular framework BioPlex 20 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization
1,122 citations
TL;DR: A review of the latest applications of CRISPR-Cas9 in mammalian functional genomics screens is presented in this article, which covers related genome-scale applications of Cas9 for either gene knockout or transcriptional modulation.
Abstract: CRISPR–Cas9 has been adopted as a powerful genome-editing technology in various species. By generating libraries of thousands of guide RNAs — which direct the Cas9 nuclease to chosen genomic loci — high-throughput genetic perturbations are now possible. This Review discusses the latest applications of CRISPR–Cas9 in mammalian functional genomics screens. It covers related genome-scale applications of Cas9 for either gene knockout or transcriptional modulation, and provides comparisons with complementary RNA interference (RNAi)-based approaches.
980 citations
References
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TL;DR: Burrows-Wheeler Alignment tool (BWA) is implemented, a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps.
Abstract: Motivation: The enormous amount of short reads generated by the new DNA sequencing technologies call for the development of fast and accurate read alignment programs. A first generation of hash table-based methods has been developed, including MAQ, which is accurate, feature rich and fast enough to align short reads from a single individual. However, MAQ does not support gapped alignment for single-end reads, which makes it unsuitable for alignment of longer reads where indels may occur frequently. The speed of MAQ is also a concern when the alignment is scaled up to the resequencing of hundreds of individuals.
Results: We implemented Burrows-Wheeler Alignment tool (BWA), a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps. BWA supports both base space reads, e.g. from Illumina sequencing machines, and color space reads from AB SOLiD machines. Evaluations on both simulated and real data suggest that BWA is ~10–20× faster than MAQ, while achieving similar accuracy. In addition, BWA outputs alignment in the new standard SAM (Sequence Alignment/Map) format. Variant calling and other downstream analyses after the alignment can be achieved with the open source SAMtools software package.
Availability: http://maq.sourceforge.net
Contact: [email protected]
43,862 citations
TL;DR: The 1000 Genomes Project aims to provide a deep characterization of human genome sequence variation as a foundation for investigating the relationship between genotype and phenotype as mentioned in this paper, and the results of the pilot phase of the project, designed to develop and compare different strategies for genomewide sequencing with high-throughput platforms.
Abstract: The 1000 Genomes Project aims to provide a deep characterization of human genome sequence variation as a foundation for investigating the relationship between genotype and phenotype. Here we present results of the pilot phase of the project, designed to develop and compare different strategies for genome-wide sequencing with high-throughput platforms. We undertook three projects: low-coverage whole-genome sequencing of 179 individuals from four populations; high-coverage sequencing of two mother-father-child trios; and exon-targeted sequencing of 697 individuals from seven populations. We describe the location, allele frequency and local haplotype structure of approximately 15 million single nucleotide polymorphisms, 1 million short insertions and deletions, and 20,000 structural variants, most of which were previously undescribed. We show that, because we have catalogued the vast majority of common variation, over 95% of the currently accessible variants found in any individual are present in this data set. On average, each person is found to carry approximately 250 to 300 loss-of-function variants in annotated genes and 50 to 100 variants previously implicated in inherited disorders. We demonstrate how these results can be used to inform association and functional studies. From the two trios, we directly estimate the rate of de novo germline base substitution mutations to be approximately 10(-8) per base pair per generation. We explore the data with regard to signatures of natural selection, and identify a marked reduction of genetic variation in the neighbourhood of genes, due to selection at linked sites. These methods and public data will support the next phase of human genetic research.
7,538 citations
5,607 citations
TL;DR: An initial version of a proteome-scale map of human binary protein–protein interactions is described, which increases by ∼70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins.
Abstract: Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.
2,936 citations
TL;DR: A screen based on high-content imaging was developed to identify genes required for mitotic progression in human cancer cells and applied to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes, providing a widely applicable resource for loss-of-function screens.
1,760 citations