High-throughput Genetic Screens Research Articles

Simple in Vivo Gene Editing via Direct Self-Assembly of Cas9 Ribonucleoprotein Complexes for Cancer Treatment.

Cas9 ribonucleoprotein (RNP)-mediated delivery has emerged as an ideal approach for in vivo applications. However, the delivery of Cas9 RNPs requires electroporation or lipid- or cationic-reagent-mediated transfection. Here, we developed a carrier-free Cas9 RNP delivery system for robust gene editing in vivo. For simultaneous delivery of Cas9 and a guide RNA into target cells without the aid of any transfection reagents, we established a multifunctional Cas9 fusion protein (Cas9-LMWP) that forms a ternary complex with synthetic crRNA:tracrRNA hybrids in a simple procedure. Cas9-LMWP carrying both a nuclear localization sequence and a low-molecular-weight protamine (LMWP) enables the direct self-assembly of a Cas9:crRNA:tracrRNA ternary complex (a ternary Cas9 RNP) and allows for the delivery of the ternary Cas9 RNPs into the recipient cells, owing to its intrinsic cellular and nuclear translocation ability with low immunogenicity. To demonstrate the potential of this system, we showed extensive synergistic anti-KRAS therapy (CI value: 0.34) via in vitro and in vivo editing of the KRAS gene by the direct delivery of multifunctional Cas9 RNPs in lung cancer. Thus, our carrier-free Cas9 RNP delivery system could be an innovative platform that might serve as an alternative to conventional transfection reagents for simple gene editing and high-throughput genetic screening.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem.

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up. Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans-Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches. We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans-E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

Abstract 1: A kinome-wide CRISPR screen reveals BET inhibition-associated endothelial cell resistance to anti-angiogenic therapy

Abstract Angiogenesis inhibition is a useful strategy for treating cancer. However, the efficacy of anti-angiogenic therapy (AAT) in clinical oncology has been limited largely by highly variable patient response and the inevitable occurrence of resistance. The modest patient benefits have underscored a pressing need for qualified biomarkers and better knowledge of resistance mechanisms. Blood endothelial cells (ECs) are one of the main targets of AAT in the tumor microenvironment. Hence, understanding the EC response to AAT will provide insight into tumor response. To identify molecular modifiers of the EC response to AAT (in this study, bevacizumab, a humanized neutralizing anti-VEGF-A monoclonal antibody), we developed a high-throughput genetic screening platform. This involved a three-dimensional microcarrier-based culture system, CRISPR-Cas9-driven gene loss-of-function (LOF) and VEGF-A-dependent serum-free culture conditions for response modifiers. A pooled kinome-wide CRISPR-Cas9-based screen of 763 genes (with four single guide RNAs/sgRNAs targeting each gene) identified 18 candidate genes that upon LOF (represented by sgRNAs) were significantly enriched or depleted in the bevacizumab versus control treatment arm (P ≤ 0.005, FDR ≤ 0.3). Further candidate evaluation using siRNAs validated six genes whose knockdown conferred EC resistance or sensitization to bevacizumab (P < 0.05). Of these, knockdown of BRD2 or BRD3, which encode the epigenetic reader bromodomain-containing protein 2 or 3 (BRD2 or BRD3), respectively, conferred EC resistance to bevacizumab. The bromodomain and extraterminal domain (BET) inhibitors JQ1 and I-BET762, which selectively target the BET family of proteins (BRD2, BRD3, BRD4 and BRDT), reproduced the results of BRD2 or BRD3 LOF, with a more prominent phenotype (P < 0.05). Drug dose-response assessment indicated an anti-angiogenic effect of BET inhibitors regardless of the presence of bevacizumab. This inhibitory effect was unexpectedly attenuated when cells were co-treated with bevacizumab under VEGF-A-dependent conditions. Experiments to investigate the mechanistic basis for this phenotype of resistance are ongoing and include differential gene expression analysis using RNA-Seq and in vivo evaluation. Applying a non-biased approach to identify molecular modifiers of the EC response to AAT, we demonstrate in this study that BET inhibition is unexpectedly associated with resistance to bevacizumab, despite BET inhibition alone having an anti-angiogenic effect. These observations prompt further evaluation of epigenetic regulation in tumor angiogenesis, particularly in the context of interaction between BET inhibition and VEGF blockade. Clinically, these findings may facilitate development of potential predictive and/or response biomarkers and strategies to overcome resistance to AAT and/or BET inhibitors. Citation Format: Michael Y. He, Michael M. Halford, Marc G. Achen, Steven A. Stacker. A kinome-wide CRISPR screen reveals BET inhibition-associated endothelial cell resistance to anti-angiogenic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1.

Mycobacterium tuberculosis Reporter Strains as Tools for Drug Discovery and Development.

Reporter strains have proven to be powerful tools to study Mycobacterium tuberculosis (Mtb) physiology. Transcriptional and translational reporter strains are engineered by fusing a readout gene, encoding a fluorescent, luminescent or enzymatic protein, downstream of a promoter or in-frame with a gene of interest. When the reporter is expressed, it generates a signal that acts as a synthetic phenotype, enabling the study of physiologies that might have otherwise been hidden. This review will discuss approaches for generating reporter strains in Mtb and how they can be used as tools for high-throughput genetic and small molecule screening and as biomarkers for examining Mtb responses to drug or immune stresses during animal infections. Fluorescent reporter strains have an added benefit in that they can be used for single-cell studies both in vitro and in vivo, thus enabling the study of mechanisms underlying phenotypic heterogeneity. Recent examples of the use of Mtb reporter strains will be presented with a focus on how they can be used as tools for drug discovery and development. © 2018 IUBMB Life, 70(9):818-825, 2018.

High-throughput genetic screens using CRISPR-Cas9 system.

The CRISPR-Cas9 system is a powerful tool for genome engineering, and its programmability and simplicity have enabled various types of gene manipulation such as gene disruption and transcriptional and epigenetic perturbation. Particularly, CRISPR-based pooled libraries facilitate high-throughput screening for functional regulatory elements in the human genome. In this review, we describe recent advances in CRISPR-Cas9 technology and its use in high-throughput genetic screening. We also discuss its potential for drug target discovery and current challenges of this technique in biomedical research.

High-throughput interaction screens illuminate the role of c-di-AMP in cyanobacterial nighttime survival.

The broadly conserved signaling nucleotide cyclic di-adenosine monophosphate (c-di-AMP) is essential for viability in most bacteria where it has been studied. However, characterization of the cellular functions and metabolism of c-di-AMP has largely been confined to the class Bacilli, limiting our functional understanding of the molecule among diverse phyla. We identified the cyclase responsible for c-di-AMP synthesis and characterized the molecule’s role in survival of darkness in the model photosynthetic cyanobacterium Synechococcus elongatus PCC 7942. In addition to the use of traditional genetic, biochemical, and proteomic approaches, we developed a high-throughput genetic interaction screen (IRB-Seq) to determine pathways where the signaling nucleotide is active. We found that in S. elongatus c-di-AMP is produced by an enzyme of the diadenylate cyclase family, CdaA, which was previously unexplored experimentally. A cdaA-null mutant experiences increased oxidative stress and death during the nighttime portion of day-night cycles, in which potassium transport is implicated. These findings suggest that c-di-AMP is biologically active in cyanobacteria and has non-canonical roles in the phylum including oxidative stress management and day-night survival. The pipeline and analysis tools for IRB-Seq developed for this study constitute a quantitative high-throughput approach for studying genetic interactions.

Implementation of the next generation sequencing based preimplantation genetic screening in clinical practice: challenges and benefits

Introduction: The success of an in vitro fertilization (IVF) is based on variety of factors, mainly blastocyst morphology and the chromosome status of the embryo as well as an adequate endometrial receptivity. The implementation of molecular genetic assays in the assisted reproduction (AR) field worldwide allows preimplantation embryo karyotype examination via preimplantation genetic screening (PGS) techniques. The focus in the PGS/PGD field has now shifted from day 3 blastomere biopsy to day 5/6 trophoectoderm (TE) sampling and utilization of comprehensive chromosome screening technology as microarray Compartive Genomic Hybridization (aCGH) or Next Generation Sequencing (NGS) in order to provide a more accurate assesment of implantation and developmental potential of embryos. The TE as biopsy material and NGS as high throughput genetic screening technique are the logical algorithm in the successful ART strategy. In the present study we report our experience on implementation these methods in the clinical practice.

Direct transfection of clonal organoids in Matrigel microbeads: a promising approach toward organoid-based genetic screens.

Organoid cultures in 3D matrices are relevant models to mimic the complex in vivo environment that supports cell physiological and pathological behaviors. For instance, 3D epithelial organoids recapitulate numerous features of glandular tissues including the development of fully differentiated acini that maintain apico-basal polarity with hollow lumen. Effective genetic engineering in organoids would bring new insights in organogenesis and carcinogenesis. However, direct 3D transfection on already formed organoids remains challenging. One limitation is that organoids are embedded in extracellular matrix and grow into compact structures that hinder transfection using traditional techniques. To address this issue, we developed an innovative approach for transgene expression in 3D organoids by combining single-cell encapsulation in Matrigel microbeads using a microfluidic device and electroporation. We demonstrate that direct electroporation of encapsulated organoids reaches up to 80% of transfection efficiency. Using this technique and a morphological read-out that recapitulate the different stages of tumor development, we further validate the role of p63 and PTEN as key genes in acinar development in breast and prostate tissues. We believe that the combination of controlled organoid generation and efficient 3D transfection developed here opens new perspectives for flow-based high-throughput genetic screening and functional genomic applications.

New Developments in CRISPR/Cas-based Functional Genomics and their Implications for Research Using Zebrafish.

Genome editing using CRISPR/Cas9 has advanced very rapidly in its scope, versatility and ease of use. Zebrafish (Danio rerio) has been one of the vertebrate model species where CRISPR/Cas9 has been applied very extensively for many different purposes and with great success. In particular, disease modeling in zebrafish is useful for testing specific gene variants for pathogenicity in a preclinical setting. Here we describe multiple advances in diverse species and systems that can improve genome editing in zebrafish. To achieve temporal and spatial precision of genome editing, many new technologies can be applied in zebrafish such as artificial transcription factors, drug-inducible or optogenetically-driven expression of Cas9, or chemically-inducible activation of Cas9. Moreover, chemically- or optogenetically- inducible reconstitution of dead Cas9 (catalytically inactive, dCas9) can enable spatiotemporal control of gene regulation. In addition to controlling where and when genome editing occurs, using oligonucleotides allows for the introduction (knock-in) of precise modifications of the genome. We review recent trends to improve the precision and efficiency of oligo-based point mutation knock-ins and discuss how these improvements can apply to work in zebrafish. Similarly to how chemical mutagenesis enabled the first genetic screens in zebrafish, multiplexed sgRNA libraries and Cas9 can enable the next revolutionary transition in how genetic screens are performed in this species. We discuss the first examples and prospects of approaches using sgRNAs as specific and effective mutagens. Moreover, we have reviewed methods aimed at measuring the phenotypes of single cells after their mutagenic perturbation with vectors encoding individual sgRNAs. These methods can range from different cell-based reporters to single-cell RNA sequencing and can serve as great tools for high-throughput genetic screens.

The Ancient Genetic Networks of Obesity: Whole-Animal Automated Screening for Conserved Fat Regulators.

Caenorhabditis elegans is the first and only metazoan model that enables whole-body gene knockdown by simply feeding their standard laboratory diet, E. coli, carrying RNA interference (RNAi)-expressing constructs. The simplicity of the RNAi treatment, small size, and fast reproduction rate of C. elegans allow us to perform whole-animal high-throughput genetic screens in wild-type, mutant, or otherwise genetically modified C. elegans. In addition, more than 65% of C. elegans genes are conserved in mammals including human. In particular, C. elegans metabolic pathways are highly conserved, which supports the study of complex diseases such as obesity in this genetically tractable model system. In this chapter, we present a detailed protocol for automated high-throughput whole-animal RNAi screening to identify the pathways promoting obesity in diet-induced and genetically driven obese C. elegans. We describe an optimized high-content screening protocol to score fat mass and body fat distribution in whole animals at large scale. We provide optimized pipelines to automatically score phenotypes using the open-source CellProfiler platform within the context of supercomputer clusters. Further, we present a guideline to optimize information workflow from the automated microscope to a searchable database. The approaches described here enable unveiling the whole network of gene-gene and gene-environment interactions that define metabolic health or disease status in this proven model of human disease, but similar principles can be applied to other disease models.

Abstract 28: Identifying mechanisms of PD-L1 regulation in HNSCC

Abstract Evidence of immunosuppression is frequently reported in head and neck squamous cell carcinoma (HNSCC). Thus, there has been growing interest in advancing therapies for HNSCC that relieving immunosuppression to permit anti-tumor immunity. The immune checkpoint inhibitor Pembrolizumab targets the coinhibitory PD-1/PD-L1 pathway, which serves to shield tumor cells from attack by cytotoxic T lymphocytes. While Pembrolizumab was recently approved for the treatment of metastatic and recurrent HNSCC, the initial trial reported overall response rates of only 18%. A more comprehensive understanding of the intrinsic and extrinsic mechanisms inducing PD-L1 expression on tumor cells may reveal therapeutic targets that improve response to immune modulation. We have employed a high-throughput genetic screen to identify targetable signaling pathways that may regulate PD-L1 expression in patients with specific genetic lesions. The Genome-scale CRISPR Knock-Out (GeCKO) library, established in multiple HNSCC cell lines, consists of a pool of stable knockouts targeting >20,000 genes with representation of approximately 300 gRNAs per target gene. Sub-populations with enhanced or diminished expression of PD-L1 were sequenced to identify gRNA targets whose knockout causes a change in PD-L1 expression. We expect that by performing these screens across multiple cell lines with diverse genetic lesions, pathways consistently regulating PD-L1 expression will be revealed, and that targeting such pathways will provide novel avenues for modulating this immunosuppressive mechanism. Citation Format: Jacqueline E. Mann, Megan Ludwig, Aditi Kulkarni, Rebecca Hoesli, Judy Kafelghazal, Alexey Nesvizhskii, J. Chad Brenner. Identifying mechanisms of PD-L1 regulation in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 28.

Germline Cas9 expression yields highly efficient genome engineering in a major worldwide disease vector, Aedes aegypti

The development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general, in vivo germline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vector Aedes aegypti Here, we describe the generation of multiple stable, transgenic Ae. aegypti strains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR. Using these strains, we disrupted numerous genes important for normal morphological development, and even generated triple mutants from a single injection. We have also managed to increase the rates of homology-directed repair by more than an order of magnitude. Given the exceptional mutagenic efficiency and specificity of the Cas9 strains we engineered, they can be used for high-throughput reverse genetic screens to help functionally annotate the Ae. aegypti genome. Additionally, these strains represent a step toward the development of novel population control technologies targeting Ae. aegypti that rely on Cas9-based gene drives.

A High Throughput Genetic Screen to Identify Causal Mediators of Variability in Drug Induced Cardiotoxicity

A High Throughput Genetic Screen to Identify Causal Mediators of Variability in Drug Induced Cardiotoxicity

High-Throughput Functional Genetic and Compound Screens Identify Targets for Senescence Induction in Cancer

Senescence is a proliferation arrest that can result from a variety of stresses. Cancer cells can also undergo senescence, but the stresses that provoke cancer cells to undergo senescence are unclear. Here, we use both functional genetic and compound screens in cancer cells harboring a reporter that is activated during senescence to find targets that induce senescence. We show that suppression of the SWI/SNF component SMARCB1 induces senescence in melanoma through strong activation of the MAP kinase pathway. From the compound screen, we identified multiple aurora kinase inhibitors as potent inducers of senescence in RAS mutant lung cancer. Senescent melanoma and lung cancer cells acquire sensitivity to the BCL2 family inhibitor ABT263. We propose a one-two punch approach for the treatment of cancer in which a drug is first used to induce senescence in cancer cells and a second drug is then used to kill senescent cancer cells.

High-Throughput Genetic Screening of 51 Pediatric Cataract Genes Identifies Causative Mutations in Inherited Pediatric Cataract in South Eastern Australia.

Pediatric cataract is a leading cause of childhood blindness. This study aimed to determine the genetic cause of pediatric cataract in Australian families by screening known disease-associated genes using massively parallel sequencing technology. We sequenced 51 previously reported pediatric cataract genes in 33 affected individuals with a family history (cases with previously known or published mutations were excluded) using the Ion Torrent Personal Genome Machine. Variants were prioritized for validation if they were predicted to alter the protein sequence and were absent or rare with minor allele frequency <1% in public databases. Confirmed mutations were assessed for segregation with the phenotype in all available family members. All identified novel or previously reported cataract-causing mutations were screened in 326 unrelated Australian controls. We detected 11 novel mutations in GJA3, GJA8, CRYAA, CRYBB2, CRYGS, CRYGA, GCNT2, CRYGA, and MIP; and three previously reported cataract-causing mutations in GJA8, CRYAA, and CRYBB2. The most commonly mutated genes were those coding for gap junctions and crystallin proteins. Including previous reports of pediatric cataract-associated mutations in our Australian cohort, known genes account for >60% of familial pediatric cataract in Australia, indicating that still more causative genes remain to be identified.

CRISPR/Cas9 Genome-Editing System in Human Stem Cells: Current Status and Future Prospects.

Genome-editing involves the insertion, deletion, or replacement of DNA in the genome of a living organism using “molecular scissors.” Traditional genome editing with engineered nucleases for human stem cells is limited by its low efficiency, high cost, and poor specificity. The CRISPR system has recently emerged as a powerful gene manipulation technique with advantages of high editing efficiency and low cost. Although this technique offers huge potential for gene manipulation in various organisms ranging from prokaryotes to higher mammals, there remain many challenges in human stem cell research. In this review, we highlight the basic biology and application of the CRISPR/Cas9 system in current human stem cell research, discuss its advantages and challenges, and debate the future prospects for human stem cells in regenerative medicine.

CRISPR-UnLOCK: Multipurpose Cas9-Based Strategies for Conversion of Yeast Libraries and Strains.

Saccharomyces cerevisiae continues to serve as a powerful model system for both basic biological research and industrial application. The development of genome-wide collections of individually manipulated strains (libraries) has allowed for high-throughput genetic screens and an emerging global view of this single-celled Eukaryote. The success of strain construction has relied on the innate ability of budding yeast to accept foreign DNA and perform homologous recombination, allowing for efficient plasmid construction (in vivo) and integration of desired sequences into the genome. The development of molecular toolkits and “integration cassettes” have provided fungal systems with a collection of strategies for tagging, deleting, or over-expressing target genes; typically, these consist of a C-terminal tag (epitope or fluorescent protein), a universal terminator sequence, and a selectable marker cassette to allow for convenient screening. However, there are logistical and technical obstacles to using these traditional genetic modules for complex strain construction (manipulation of many genomic targets in a single cell) or for the generation of entire genome-wide libraries. The recent introduction of the CRISPR/Cas gene editing technology has provided a powerful methodology for multiplexed editing in many biological systems including yeast. We have developed four distinct uses of the CRISPR biotechnology to generate yeast strains that utilizes the conversion of existing, commonly-used yeast libraries or strains. We present Cas9-based, marker-less methodologies for (i) N-terminal tagging, (ii) C-terminally tagging yeast genes with 18 unique fusions, (iii) conversion of fluorescently-tagged strains into newly engineered (or codon optimized) variants, and finally, (iv) use of a Cas9 “gene drive” system to rapidly achieve a homozygous state for a hypomorphic query allele in a diploid strain. These CRISPR-based methods demonstrate use of targeting universal sequences previously introduced into a genome.

High-Throughput Genetic Screen Reveals that Early Attachment and Biofilm Formation Are Necessary for Full Pyoverdine Production by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a re-emerging, multidrug-resistant, opportunistic pathogen that threatens the lives of immunocompromised patients, patients with cystic fibrosis, and those in critical care units. One of the most important virulence factors in this pathogen is the siderophore pyoverdine. Pyoverdine serves several critical roles during infection. Due to its extremely high affinity for ferric iron, pyoverdine gives the pathogen a significant advantage over the host in their competition for iron. In addition, pyoverdine can regulate the production of multiple bacterial virulence factors and perturb host mitochondrial homeostasis. Inhibition of pyoverdine biosynthesis decreases P. aeruginosa pathogenicity in multiple host models. To better understand the regulation of pyoverdine production, we developed a high-throughput genetic screen that uses the innate fluorescence of pyoverdine to identify genes necessary for its biosynthesis. A substantial number of hits showing severe impairment of pyoverdine production were in genes responsible for early attachment and biofilm formation. In addition to genetic disruption of biofilm, both physical and chemical perturbations also attenuated pyoverdine production. This regulatory relationship between pyoverdine and biofilm is particularly significant in the context of P. aeruginosa multidrug resistance, where the formation of biofilm is a key mechanism preventing access to antimicrobials and the immune system. Furthermore, we demonstrate that the biofilm inhibitor 2-amino-5,6-dimethylbenzimidazole effectively attenuates pyoverdine production and rescues Caenorhabditis elegans from P. aeruginosa-mediated pathogenesis. Our findings suggest that targeting biofilm formation in P. aeruginosa infections may have multiple therapeutic benefits and that employing an unbiased, systems biology-based approach may be useful for understanding the regulation of specific virulence factors and identifying novel anti-virulence therapeutics or new applications for existing therapies for P. aeruginosa infections.

Characterization of noncoding regulatory DNA in the human genome.

Genetic variants associated with common diseases are usually located in noncoding parts of the human genome. Delineation of the full repertoire of functional noncoding elements, together with efficient methods for probing their biological roles, is therefore of crucial importance. Over the past decade, DNA accessibility and various epigenetic modifications have been associated with regulatory functions. Mapping these features across the genome has enabled researchers to begin to document the full complement of putative regulatory elements. High-throughput reporter assays to probe the functions of regulatory regions have also been developed but these methods separate putative regulatory elements from the chromosome so that any effects of chromatin context and long-range regulatory interactions are lost. Definitive assignment of function(s) to putative cis-regulatory elements requires perturbation of these elements. Genome-editing technologies are now transforming our ability to perturb regulatory elements across entire genomes. Interpretation of high-throughput genetic screens that incorporate genome editors might enable the construction of an unbiased map of functional noncoding elements in the human genome.

Abstract 402: Biomarker-based pooled CRISPR-Cas9 screening: Platform development and validation

Abstract Functional genomic screening with CRISPR-Cas9 has provided a powerful and precise new way to interrogate the phenotypic consequences of gene loss in high-throughput, unbiased analyses. Rapid development of pooled lentivirus and deep-sequencing-led approaches have allowed us and others to exploit this technology in target ID, target validation, drug MOA analysis and patient stratification. Adaptation of the Cas9 nuclease by domain fusion and catalytic inactivation has further provided a novel way to both up and down-regulate gene expression using targeted, high-throughput CRISPRi and CRISPRa tools. To date, the vast majority of screens have been conducted using sgRNA abundance in surviving cell populations as an NGS-based proxy for proliferation, for example for in vitro resistance analysis to a cytotoxic treatment regimen. However, where cellular pathophysiology is uncoupled from cell health, the enormous promise of pooled CRISPR-Cas9 screening has been largely untapped. We now report on the development of a FACS-based platform that can be used to monitor biomarker-based cellular response in combination with high-throughput genetic screening. The endogenous interaction between CD28 and CD80 was used as a proof-of-concept paradigm and the performance of our platform was measured in screen-simulation conditions. We find that FACS-linked NGS was able to robustly identify the CD28 deleted cells from a complex population using a FITC-labelled CD80-Fc. Pseudo-blinding of the data yielded unambiguous identification of the expected target guides, with robust screening metrics, validating a new platform for myriad discovery programmes. Citation Format: Tim M. Scales, Benedict C. Cross. Biomarker-based pooled CRISPR-Cas9 screening: Platform development and validation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 402. doi:10.1158/1538-7445.AM2017-402