Pooled optical screens in human cells

Large-scale genetic screens play a key role in the systematic discovery of genes underlying cellular phenotypes. Pooling of genetic perturbations greatly increases screening throughput, but has so far been limited to screens of enrichments defined by cell fitness and flow cytometry, or to comparatively low-throughput single cell gene expression profiles. Although microscopy is a rich source of spatial and temporal information about mammalian cells, high-content imaging screens have been restricted to much less efficient arrayed formats. Here, we introduce an optical method to link perturbations and their phenotypic outcomes at the singlecell level in a pooled setting. Barcoded perturbations are read out by targeted in situ sequencing following image-based phenotyping. We apply this technology to screen a focused set of 952 genes across >3 million cells for involvement in NF-κB activation by imaging the translocation of RelA (p65) to the nucleus, recovering 20 known pathway components and 3 novel candidate positive regulators of IL-1β and TNFα-stimulated immune responses.

[1]  Christian V. Forst,et al.  Image-Based Genome-Wide siRNA Screen Identifies Selective Autophagy Factors , 2011, Nature.

[2]  Daniel Becker,et al.  Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication , 2010, Nature.

[3]  Chun Jimmie Ye,et al.  A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks , 2015, Cell.

[4]  George Emanuel,et al.  High-Throughput Image-Based Screening of Pooled Genetic Variant Libraries , 2019 .

[5]  André F. Rendeiro,et al.  Pooled CRISPR screening with single-cell transcriptome read-out , 2017, Nature Methods.

[6]  R. Durbin,et al.  Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes , 2010, Nature.

[7]  V. Dixit,et al.  Association of C-Terminal Ubiquitin Hydrolase BRCA1-Associated Protein 1 with Cell Cycle Regulator Host Cell Factor 1 , 2009, Molecular and Cellular Biology.

[8]  N. D. Clarke,et al.  A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity , 2010, Nature.

[9]  T. Mikkelsen,et al.  Rapid dissection and model-based optimization of inducible enhancers in human cells using a massively parallel reporter assay , 2012, Nature Biotechnology.

[10]  Timur Zhiyentayev,et al.  Single-cell in situ RNA profiling by sequential hybridization , 2014, Nature Methods.

[11]  Thomas M. Norman,et al.  A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response , 2016, Cell.

[12]  Susumu Goto,et al.  KEGG: Kyoto Encyclopedia of Genes and Genomes , 2000, Nucleic Acids Res..

[13]  William E. Allen,et al.  Three-dimensional intact-tissue sequencing of single-cell transcriptional states , 2018, Science.

[14]  S. Pastorino,et al.  Germline BAP1 mutations induce a Warburg effect , 2017, Cell Death and Differentiation.

[15]  Anindya Dutta,et al.  The Deubiquitinating Enzyme BAP1 Regulates Cell Growth via Interaction with HCF-1* , 2009, The Journal of Biological Chemistry.

[16]  Wei Li,et al.  BAP1 inhibits the ER stress gene regulatory network and modulates metabolic stress response , 2017, Proceedings of the National Academy of Sciences.

[17]  Paul C. Blainey,et al.  Lentiviral co-packaging mitigates the effects of intermolecular recombination and multiple integrations in pooled genetic screens , 2018, bioRxiv.

[18]  Anne E Carpenter,et al.  A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-Content Screen , 2006, Cell.

[19]  Carolina Wählby,et al.  In situ sequencing for RNA analysis in preserved tissue and cells , 2013, Nature Methods.

[20]  Zhijian J. Chen.  Ubiquitin signalling in the NF-κB pathway , 2005, Nature Cell Biology.

[21]  S. Strittmatter,et al.  An Unbiased Expression Screen for Synaptogenic Proteins Identifies the LRRTM Protein Family as Synaptic Organizers , 2009, Neuron.

[22]  S. Mahajan,et al.  Herpes Simplex Virus Transactivator VP16 Discriminates between HCF-1 and a Novel Family Member, HCF-2 , 1999, Journal of Virology.

[23]  Jimmy Larsson,et al.  In situ genotyping of a pooled strain library after characterizing complex phenotypes , 2017, Molecular systems biology.

[24]  Ola Söderberg,et al.  In situ detection and genotyping of individual mRNA molecules , 2010, Nature Methods.

[25]  Thomas M. Norman,et al.  Approaches to maximize sgRNA-barcode coupling in Perturb-seq screens , 2018, bioRxiv.

[26]  Jay Shendure,et al.  Accurate gene synthesis with tag-directed retrieval of sequence-verified DNA molecules , 2012, Nature Methods.

[27]  Neville E. Sanjana,et al.  Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells , 2014, Science.

[28]  Thomas M Green,et al.  A public genome-scale lentiviral expression library of human ORFs , 2011, Nature Methods.

[29]  E. Lander,et al.  Genetic Screens in Human Cells Using the CRISPR-Cas9 System , 2013, Science.

[30]  Y. Kalaidzidis,et al.  Systems survey of endocytosis by multiparametric image analysis , 2010, Nature.

[31]  Tilo Buschmann,et al.  Levenshtein error-correcting barcodes for multiplexed DNA sequencing , 2013, BMC Bioinformatics.

[32]  Anne E Carpenter,et al.  The Bromodomain Protein Brd4 Insulates Chromatin from DNA Damage Signaling , 2013, Nature.

[33]  Zhijian J. Chen.  Ubiquitin signalling in the NF-kappaB pathway. , 2005, Nature cell biology.

[34]  Thomas M. Norman,et al.  Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens , 2016, Cell.

[35]  André F. Rendeiro,et al.  Pooled CRISPR screening with single-cell transcriptome read-out , 2016, bioRxiv.

[36]  I. Amit,et al.  Dissecting Immune Circuits by Linking CRISPR-Pooled Screens with Single-Cell RNA-Seq , 2016, Cell.

[37]  Robert V Farese,et al.  Functional genomic screen reveals genes involved in lipid-droplet formation and utilization , 2008, Nature.

[38]  H. Pahl.  Activators and target genes of Rel/NF-κB transcription factors , 1999, Oncogene.

[39]  Nico Stuurman,et al.  Computer Control of Microscopes Using µManager , 2010, Current protocols in molecular biology.

[40]  X. Zhuang,et al.  Spatially resolved, highly multiplexed RNA profiling in single cells , 2015, Science.

[41]  John Quackenbush,et al.  Genome-wide siRNA screen for mediators of NF-κB activation , 2012, Proceedings of the National Academy of Sciences.

[42]  G. Hon,et al.  Frequent sgRNA-barcode recombination in single-cell perturbation assays , 2018, bioRxiv.

[43]  Neville E. Sanjana,et al.  Improved vectors and genome-wide libraries for CRISPR screening , 2014, Nature Methods.

[44]  G. Church,et al.  Efficient in situ barcode sequencing using padlock probe-based BaristaSeq , 2017, bioRxiv.

[45]  Qikai Xu,et al.  Sources of Error in Mammalian Genetic Screens , 2016, G3: Genes, Genomes, Genetics.

[46]  George M. Church,et al.  Highly Multiplexed Subcellular RNA Sequencing in Situ , 2014, Science.

[47]  Cole Trapnell,et al.  On the design of CRISPR-based single cell molecular screens , 2018, Nature Methods.