sgRNA Library Research Articles

Abstract 5945: CRISPR-Cas9 dissection of heparan sulfate

Abstract Heparan sulfate proteoglycans (HSPGs) are expressed on virtually all animal cells and play important roles in tumor growth and metastasis. Each HSPG consists of a core protein with one or more covalently attached linear heparan sulfate (HS) chains composed of alternating glucosamine and uronic acids that are heterogeneously N- and O-sulfated. These complex cell surface carbohydrates regulate important biological processes including cell proliferation and development through their interaction with a large number of matrix proteins and growth factors. The arrangement and orientation of the sulfated sugar residues of HS specify the location of distinct ligand binding sites on the cell surface, and these modifications can vary temporally during development and spatially across tissues. Previous studies have shown that HSPGs can modulate tumor growth kinetics and are abundantly expressed on the cell surface of many types of cancer. Additionally, genetically reducing the sulfation of HS has been shown to selectively inhibit tumor angiogenesis and lymph node metastasis. While most of the enzymes involved in HS biosynthesis have been studied extensively, much less information exists regarding the specific mechanisms that give rise to the variable composition and binding properties of HS. The overall goal of this project is to uncover and characterize novel genes whose expression influences HS-mediated regulatory networks in tumor growth and metastasis. A genome-wide CRISPR/Cas9-mediated screen was developed to uncover and characterize novel genes other than those encoding known HS biosynthetic enzymes. A lentiviral single guide RNA (sgRNA) library was utilized to knock down gene expression across the entire genome in a human malignant melanoma cell line. Subsequently, a high-throughput screening assay was adapted to identify lentiviral-encoded sgRNAs that induce resistance to cytotoxins whose action depends on HSPGs. Parallel screens using alternative HS-dependent toxins or plant lectins that cause cytotoxicity dependent on other types of glycosylation were performed in order to sort genes that selectively affect HS biosynthesis. From the toxin screens, we identified previously studied genes essential for HS formation and factors involved in the intoxication of cells by diphtheria toxin, an HS-dependent exotoxin. Furthermore, we uncovered potential candidate genes whose function is unknown relative to HS biosynthesis. Top hits from the screens were characterized and categorized based on their predicted gene functions and are currently being individually validated and examined for their potential involvement in the regulation of HS biosynthesis. Overall, these studies will provide a better understanding of the genetic regulatory factors involved in HS biogenesis. Additionally, the factors we identify could reveal novel targets for anti-cancer therapies, as well as lead us to methods to manipulate HS and its activities in other cellular processes that go awry in human diseases. Citation Format: Ryan J. Weiss, Philipp N. Spahn, Nathan E. Lewis, Jeffrey D. Esko. CRISPR-Cas9 dissection of heparan sulfate [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 5945. doi:10.1158/1538-7445.AM2017-5945

Abstract 3142: Aberrantly expressed microRNAs drive the development of acquired Erlotinib resistance in non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer-related deaths in the world. Non-small cell lung cancer (NSCLC) accounts for ~85% of the cases. NSCLC patients frequently harbor causal gene mutations. Epidermal Growth Factor Receptor (EGFR), an NSCLC causal gene, is mutated in 10-35% of NSCLC patients. Patients with EGFR activating mutation are treated with a tyrosine-kinase inhibitor, Erlotinib, specifically targeting EGFR. However, most patients develop Erlotinib resistance within a year. Although multiple mechanisms are involved in the development of Erlotinib resistance, the role of microRNAs in mediating Erlotinib resistance is largely unexplored. MicroRNAs (miRNAs) are small non-coding RNAs that regulate normal cellular physiology. In cancers, miRNAs are severely dysregulated, contributing to multiple cancer processes, including drug response. Despite the involvement of miRNAs in cancer, their direct role as drivers of drug resistance remain understudied, therefore, there is a critical need to understand the role of miRNAs in inducing Erlotinib resistance. To this end, we propose an unbiased two-prong approach to identify the miRNAs that drive the development of Erlotinib resistance. Therefore, miRNAs are either being (i) overexpressed, or (ii) silenced in Erlotinib sensitive cells with the hypothesis that perturbed miRNA levels will drive Erlotinib resistance. Erlotinib sensitive NSCLC cell lines, EKVX and H322M were identified from the NCI-60 Developmental Therapeutics Project and their Erlotinib dose response curves were established. To perform the miRNA overexpression screen, EKVX and H322M cells stably expressing renilla and firefly luciferase genes were generated, which will be used to monitor cell number and transfection efficiency, respectively. The luciferase-expressing cells will be transfected with a library of 2,019 individually arrayed human miRNAs and cell growth in the presence of Erlotinib will be monitored. The second prong of the study will identify miRNAs, that when lost, confer Erlotinib resistance. 400-fold coverage of small guide RNA (sgRNA) library of the CRISPR-Cas9 system was transduced in Cas9 overexpressing EKVX cells, to knockout ~21,000 human encoded genes (~1800 miRNA genes). Cells are being cultured in the presence of 75% or 90% growth inhibitory concentrations (GI75 or GI90) of Erlotinib so that only cells with sgRNAs against genes critical for Erlotinib response, survive and grow due to acquired resistance. DNA from these cells will be harvested, the sgRNAs sequenced, and compared to the sgRNAs present in Erlotinib-untreated cells. Successful completion of this project will identify miRNAs that drive Erlotinib resistance and may contribute towards development of miRNA therapeutics to enhance Erlotinib sensitivity of NSCLC tumors. Citation Format: Arpita S. Pal, Alejandra Agredo, Andrea L. Kasinski. Aberrantly expressed microRNAs drive the development of acquired Erlotinib resistance in non-small cell lung cancer [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 3142. doi:10.1158/1538-7445.AM2017-3142

Abstract 401: Designing for success: the right CRISPR design strategies for the right experiment

Abstract CRISPR design is a complex multivariate problem. The weight of each variable differs depending on the experiment under consideration. Current methods of designing CRISPR experiments are based on a single-variable, resulting in poor experimental outcomes. From the numerous experiments enabled by the DESKGEN platform, we can demonstrate key design improvements to maximise experimental outcome. Our current scoring methods were tested and validated using an sgRNA library of over 3,500 sgRNAs in essential genes with a variety of predicted sgRNA activities and specificities. The predicted 'good' sgRNAs achieved significantly higher read depletion for essential genes, with fewer failing sgRNAs, compared to sgRNAs that didn't meet the scoring criteria. Our scoring algorithm incorporates multiple published observations for choosing effective sgRNAs, which differs from other CRISPR tools that tend to focus on one factor such as sgRNA sequence or specificity. We were unable to find correlations for some currently accepted sgRNA selection criteria, such as position within a gene, which demonstrates the need to constantly challenge and improve sgRNA design rules. Citation Format: Leigh Brody, Neil Humphryes-Kirilov, Victor Dillard, Riley Doyle. Designing for success: the right CRISPR design strategies for the right experiment [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 401. doi:10.1158/1538-7445.AM2017-401

Abstract LB-118: Characterization of lineage vs. context-dependent essential genes in multiple myeloma using CRISPR-Cas9 genome editing

Abstract Multiple Myeloma (MM) remains incurable in part due to an incomplete understanding of the genes critically responsible for MM cell survival and proliferation. We reasoned that CRISPR/Cas9-based functional genomics could identify genes essential for survival or proliferation of MM cells and define candidate therapeutic targets. We transduced Cas9-expressing RPMI-8226 and MM.1S cells with lentiviral genome-scale GeCKO pooled libraries of sgRNAs. After culture of these cell lines for 2-12 weeks without treatment we identified, based on next generation sequencing, genes with significantly depleted sgRNAs (4-6 sgRNAs/gene, >2-fold average depletion, FDR<0.05, using MAGECK algorithm) in Cas9+ cells compared to initial sgRNA plasmid pools, baseline cultures, or isogenic parental Cas9-negative cells. These results were confirmed for each cell line with a 2nd independent genome-wide analysis and with a focused sgRNA library containing a subset of candidates defined by the genome-wide analyses. We compared these results with data from our in-house or publicly available CRISPR/Cas9 gene editing studies, involving a total of 175 cell lines from other hematologic malignancies and solid tumors. We next integrated our functional data for these genes with their: a) Achilles Heel shRNA data; b) gene expression (RNASeq); c) gene copy number status; d) mutation status; e) proximity to superenhancers; and f) altered expression in MM cells co-cultured with bone marrow stromal cells. We applied a series of statistical tests (e.g. Wilcoxon rank) to identify genes with significantly higher rank in sgRNA or shRNA depletion in MM vs. non-MM lines and stringently identified 50+ candidate genes with preferential dependency in MM. Prominent examples included: transcription factors (e.g. IRF4, CCND2, MAF, NFKB1, NFKB2, RELA, RELB); other NF-κB-related genes (e.g. IKBKB); PIM2; regulators of protein homeostasis; and several other known or biologically plausible dependencies which are under further evaluation. In >90% of MM patients, the candidate MM-preferential essential genes are not mutated. Some of these genes are overexpressed in MM vs. non-MM cell lines (CCLE dataset) or patient-derived samples (Broad/MMRF vs. TCGA datasets); induced by BMSC co-culture; proximal to superenhancers; or correlating with clinical outcome. However, these molecular results were not sufficient, collectively or individually, to identify this cohort of MM-preferential essential genes independently of the CRISPR functional data. Targeting lineage-specific dependencies (e.g. ER or AR in breast or prostate Ca, respectively) has provided major clinical benefit in some tumors; while context-specific dependencies are a cornerstone of molecularly-guided individualized treatments. Therefore, by identifying lineage- and context-dependent essential genes for MM, our functional genomic studies in molecularly annotated MM vs. non-MM cell lines provide an attractive framework towards designing novel therapies for MM. Citation Format: Geoffrey M. Matthews, Ricardo de Matos Simoes, Yiguo Hu, Michal Sheffer, Olga Dashevsky, Eugen Dhimolea, Paul J. Hengeveld, Brian J. Glassner, Sara Gandolfi, Megan A. Bariteau, Quinlan Sievers, Benjamin L. Ebert, Franciska Vazquez, Aedin Culhane, Constantine S. Mitsiades. Characterization of lineage vs. context-dependent essential genes in multiple myeloma using CRISPR-Cas9 genome editing [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 LB-118. doi:10.1158/1538-7445.AM2017-LB-118

Abstract 2844: Identification of genes associated with pancreatic cancer metastasis by genome-wide CRISPR Cas9 screening

Abstract Pancreatic cancer is an extremely lethal cancer with a 5-year survival rate of 7%. One major reason why pancreatic cancer is hard to treat is its metastasis nature. Genes that related to the tumorigenesis and progression of pancreatic cancer were identified after decades of efforts. Several researches had revealed that pancreatic cancer originated from the successive accumulation of gene mutations, especially KRAS2, CNKD2A, TP53 and SMAD4. However, little is known about genes that regulate pancreatic cancer metastasis. Here we used a genome wide CRISPR Cas9 screening to identify the gene(s) associated with the metastasis of pancreatic cancer. We transduced pancreatic cancer cell line with lentivirus-based genome wide CRISPR sgRNA library to establish mutated cell library, and orthotopically injected the cell library into nude mice and waited for primary tumor growth and metastasis, then collected primary and metastatic tumors after several months. By next generation sequencing and analysis, we identified some gene candidates which may play important roles on pancreatic cancer metastasis. Note: This abstract was not presented at the meeting. Citation Format: Youjia Li, Huiyi Feng, Guangjie Liu, Chi Hin Wong, Chi Han Li, Yangchao Chen. Identification of genes associated with pancreatic cancer metastasis by genome-wide CRISPR Cas9 screening [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 2844. doi:10.1158/1538-7445.AM2017-2844

CRISPR/Cas9-mediated gene knockout screens and target identification via whole-genome sequencing uncover host genes required for picornavirus infection

Several groups have used genome-wide libraries of lentiviruses encoding small guide RNAs (sgRNAs) for genetic screens. In most cases, sgRNA expression cassettes are integrated into cells by using lentiviruses, and target genes are statistically estimated by the readout of sgRNA sequences after targeted sequencing. We present a new virus-free method for human gene knockout screens using a genome-wide library of CRISPR/Cas9 sgRNAs based on plasmids and target gene identification via whole-genome sequencing (WGS) confirmation of authentic mutations rather than statistical estimation through targeted amplicon sequencing. We used 30,840 pairs of individually synthesized oligonucleotides to construct the genome-scale sgRNA library, collectively targeting 10,280 human genes (i.e. three sgRNAs per gene). These plasmid libraries were co-transfected with a Cas9-expression plasmid into human cells, which were then treated with cytotoxic drugs or viruses. Only cells lacking key factors essential for cytotoxic drug metabolism or viral infection were able to survive. Genomic DNA isolated from cells that survived these challenges was subjected to WGS to directly identify CRISPR/Cas9-mediated causal mutations essential for cell survival. With this approach, we were able to identify known and novel genes essential for viral infection in human cells. We propose that genome-wide sgRNA screens based on plasmids coupled with WGS are powerful tools for forward genetics studies and drug target discovery.

SgRNA Scorer 2.0: A Species-Independent Model To Predict CRISPR/Cas9 Activity.

It has been possible to create tools to predict single guide RNA (sgRNA) activity in the CRISPR/Cas9 system derived from Streptococcus pyogenes due to the large amount of data that has been generated in sgRNA library screens. However, with the discovery of additional CRISPR systems from different bacteria, which show potent activity in eukaryotic cells, the approach of generating large data sets for each of these systems to predict their activity is not tractable. Here, we present a new guide RNA tool that can predict sgRNA activity across multiple CRISPR systems. In addition to predicting activity for Cas9 from S. pyogenes and Streptococcus thermophilus CRISPR1, we experimentally demonstrate that our algorithm can predict activity for Cas9 from Staphylococcus aureus and S. thermophilus CRISPR3. We also have made available a new version of our software, sgRNA Scorer 2.0, which will allow users to identify sgRNA sites for any PAM sequence of interest.

Dense sgRNA Library Construction Using a Molecular Chipper Approach.

Genetic screens using single-guide-RNA (sgRNA) libraries and CRISPR technology have been powerful to identify genetic regulators for both coding and noncoding regions of the genome. Interrogating functional elements in noncoding regions requires sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for customization. We present a Molecular Chipper protocol for generating dense sgRNA libraries from genomic regions of interest. This approach utilizes a combination of random fragmentation and a Type III restriction enzyme to derive a dense coverage of sgRNA library from input DNA.

Genome-Wide CRISPR/Cas9 Screening for High-Throughput Functional Genomics in Human Cells.

It is highly desirable to identify gene's function in a high-throughput fashion, and the CRISPR/Cas9 system has been harnessed to meet such a need. Here, we describe a general method to generate genome-scale lentiviral single-guide RNA (sgRNA) library and conduct a pooled function-based screening in human cells. This protocol would be of interest to researchers to rapidly identify genes in a variety of biological processes.

Using CRISPR-ERA Webserver for sgRNA Design.

The CRISPR-Cas9 system is emerging as a powerful technology for gene editing (modifying the genome sequence) and gene regulation (without modifying the genome sequence). Designing sgRNAs for specific genes or regions of interest is indispensable to CRISPR-based applications. CRISPR-ERA (http://crispr-era.stanford.edu/) is one of the state-of-the-art designer webserver tools, which has been developed both for gene editing and gene regulation sgRNA design. This protocol discusses how to design sgRNA sequences and genome-wide sgRNA library using CRISPR-ERA.

CRISPR Libraries and Screening.

CRISPR-Cas9 technology has revolutionized large-scale functional genomic screening in mammalian cell-culture systems. Due in part to optimized lentiviral delivery vectors; it is now possible to perform CRISPR-Cas9 screens in animals in order to study biological processes in the context of a whole organism and within more physiologically relevant environment. This chapter focuses primarily on mouse models of human cancers; viral vectors used for simultaneous tumor initiation and genome editing and sgRNA library design considerations. Experience with direct and indirect in vivo RNAi screens in the literature is also discussed in order to highlight the challenges of delivering diverse libraries of small RNAs in vivo.

Genome-Wide CRISPR Screen Identifies Regulators of Mitogen-Activated Protein Kinase as Suppressors of Liver Tumors in Mice

It has been a challenge to identify liver tumor suppressors or oncogenes due to the genetic heterogeneity of these tumors. We performed a genome-wide screen to identify suppressors of liver tumor formation in mice, using CRISPR-mediated genome editing. We performed a genome-wide CRISPR/Cas9-based knockout screen of P53-null mouse embryonic liver progenitor cells that overexpressed MYC. We infected p53-/-;Myc;Cas9 hepatocytes with the mGeCKOa lentiviral library of 67,000 single-guide RNAs (sgRNAs), targeting 20,611 mouse genes, and transplanted the transduced cells subcutaneously into nude mice. Within 1 month, all the mice that received the sgRNA library developed subcutaneous tumors. We performed high-throughput sequencing of tumor DNA and identified sgRNAs increased at least 8-fold compared to the initial cell pool. To validate the top 10 candidate tumor suppressors from this screen, we collected data from patients with hepatocellular carcinoma (HCC) using the Cancer Genome Atlas and COSMIC databases. We used CRISPR to inactivate candidate tumor suppressor genes in p53-/-;Myc;Cas9 cells and transplanted them subcutaneously into nude mice; tumor formation was monitored and tumors were analyzed by histology and immunohistochemistry. Mice with liver-specific disruption of p53 were given hydrodynamic tail-vein injections of plasmids encoding Myc and sgRNA/Cas9 designed to disrupt candidate tumor suppressors; growth of tumors and metastases was monitored. We compared gene expression profiles of liver cells with vs without tumor suppressor gene disrupted by sgRNA/Cas9. Genes found to be up-regulated after tumor suppressor loss were examined in liver cancer cell lines; their expression was knocked down using small hairpin RNAs, and tumor growth was examined in nude mice. Effects of the MEK inhibitors AZD6244, U0126, and trametinib, or the multi-kinase inhibitor sorafenib, were examined in human and mouse HCC cell lines. We identified 4 candidate liver tumor suppressor genes not previously associated with liver cancer (Nf1, Plxnb1, Flrt2, and B9d1). CRISPR-mediated knockout of Nf1, a negative regulator of RAS, accelerated liver tumor formation in mice. Loss of Nf1 or activation of RAS up-regulated the liver progenitor cell markers HMGA2 and SOX9. RAS pathway inhibitors suppressed the activation of the Hmga2 and Sox9 genes that resulted from loss of Nf1 or oncogenic activation of RAS. Knockdown of HMGA2 delayed formation of xenograft tumors from cells that expressed oncogenic RAS. In human HCCs, low levels of NF1 messenger RNA or high levels of HMGA2 messenger RNA were associated with shorter patient survival time. Liver cancer cells with inactivation of Plxnb1, Flrt2, and B9d1 formed more tumors in mice and had increased levels of mitogen-activated protein kinase phosphorylation. Using a CRISPR-based strategy, we identified Nf1, Plxnb1, Flrt2, and B9d1 as suppressors of liver tumor formation. We validated the observation that RAS signaling, via mitogen-activated protein kinase, contributes to formation of liver tumors in mice. We associated decreased levels of NF1 and increased levels of its downstream protein HMGA2 with survival times of patients with HCC. Strategies to inhibit or reduce HMGA2 might be developed to treat patients with liver cancer.

In Vivo Genome-Wide Crispr Library Screen in a Xenograft Mouse Model of Tumor Growth and Metastasis of Multiple Myeloma

Introduction: Recent data show that Multiple Myeloma (MM) always progresses from a precursor state (monoclonal gammopathy of undetermined significance [MGUS]/smoldering multiple myeloma [SMM]) to overt MM indicating that there is continuous dissemination/clonal evolution of tumor cells from the original stages of tumor development to the time of clinical presentation. A major challenge in understanding the progression and metastasis of MM is to distinguish alterations driving the tumor growth and evolution from passenger mutations. Genetic screens are powerful tools for assaying phenotypes and identifying causal genes in various hallmarks of cancer progression. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged as a powerful technology to efficiently and simultaneously perform genome editing of multiple genes. Here we report a genome-wide CRISPR/Cas9-mediated loss of function screen in a xenograft mouse model to investigate the essential drivers of tumor growth and metastasis in MM.Methods: Lentiviral particles from 2 subpools of a human sgRNA library (Avana), each containing 1 sgRNA per gene were introduced into MM1.S (Cas9+/GFP+/Luc+) cell line with the pre-determined amount of virus to achieve 30-50% infection efficiency, corresponding to a multiplicity of infection (MOI) of ~0.5-1. Cells were selected with puromycin for 5-7 days following infection to remove uninfected cells. Selected cells were injected subcutaneously into SCID-Beige mice on both flanks. Genomic DNA from pre-transplantation cells, early primary tumors (~3 weeks post tumor cell injection), late stage primary tumors and metastatic bone marrow samples were extracted. gDNA was amplified following adaptor ligation and barcoding of the samples and PCR products were subsequently sequenced on a HiSeq2000 (Illumina).Results: To investigate the sgRNA library dynamics in different sample types (pre-transplantation cells, early primary tumor, late primary tumor, and bone marrow metastasis), we compared the overall distributions of sgRNAs from all sequenced samples. The early tumor sample replicates of both subpools on average retained 77.3% and 94.7% of the sgRNAs found in the pre-transplanted cell populations, while the late primary tumors retained 59.4% and 65.6% of the sgRNAs respectively, compared to early tumors. Interestingly, only a small fraction of sgRNAs (1.1% and 3.4% of sgRNAs in the pre-transplantation cells, 10.7% and 7.2% of sgRNAs in the late primary tumors for the 2 subpools respectively) were detected in the metastatic bone marrow samples. Using gene set enrichment analysis (GSEA), we found that the gene targets of the most enriched sgRNAs in the bone marrow samples were preferentially involved in important cellular processes, such as cell cycle regulation, protein translation, and several signaling pathways. Additionally we compared sgRNAs present in early primary tumor versus pre-transplantation cells and late primary tumor and found that many sgRNAs were depleted during tumor progression, indicating that their target genes were important for progression. These depleted sgRNAs in both stages mainly targeted genes involved in mTORC1 and DNA repair pathways, many of which are regulated by MYC and cell cycle related targets of E2F transcription factors.Conclusion: We established a platform for future in vivo Cas9 screens using the genome-wide CRISPR screening libraries to explore potential new targets in regulating tumor dissemination, colonization and metastasis in MM. In addition, this in vivo screening could potentially be used to investigate essential genes of response to targeted therapies or/and immunotherapies. Thus, CRISPR/Cas9-based in vivo screening is a powerful tool for functional genomics discoveries. DisclosuresRoccaro:Takeda Pharmaceutical Company Limited: Honoraria. Ghobrial:BMS: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria; Takeda: Honoraria; Noxxon: Honoraria; Amgen: Honoraria.

Characterization of Lineage Vs. Context-Dependent Essential Genes in Multiple Myeloma Using Crispr/Cas9 Genome Editing

Multiple Myeloma (MM) remains an incurable malignancy in part because of an incomplete understanding of which genes are critically responsible for MM cell survival and proliferation. To address this unmet need, and building on our recent functional genomics studies with the CRISPR/Cas9 gene editing platform (ASH 2015; Int. MM Workshop, Rome 2015), we reasoned that quantification of sgRNA depletion in the absence of any treatment could identify genes essential for the survival or proliferation of MM cells and better define their role as candidate therapeutic targets. To this end, we transduced Cas9-expressing RPMI-8226 and MM.1S cells with the lentiviral genome-scale GeCKO pooled library of sgRNAs. After culture of these cell lines for 2, 6, 8 or 12 weeks without any treatment, we identified, based on next generation sequencing for the sgRNA sequences, genes with significantly depleted sgRNAs (4-6 sgRNAs/gene, >2-fold average depletion, FDR=0.05, based on MAGECK algorithm) in Cas9+ cells compared to their initial sgRNA plasmid pools, baseline cultures, or isogenic parental Cas9-negative cells. These results were confirmed for each cell line with a 2nd independent genome-wide analysis and with a focused sgRNA library containing a subset of candidates defined by the genome-wide analyses. We compared these results with data from our in-house or publicly available CRISPR/Cas9 gene editing studies, involving a total of 50 cell lines from other hematologic malignancies (leukemia, lymphoma) and from 8 different types of solid tumors. We identified 3 broad categories of essential genes in MM cells: a) core essential genes, with sgRNA depletion across the majority of MM and non-MM lines of our study, representing cellular processes critical for practically all lineages (e.g. genes involved in regulation of basic transcription factor complexes, ribosomal function, proteasome, spliceosome, structural proteins for mitochondria and other key organelles, et.c.); b) genes selectively essential for MM cell lines, but not for the overwhelming majority of leukemia, lymphoma or solid tumor cell lines; c) genes with a role in small subset(s) of cell lines, across diseases, which harbor defined genetic features correlating with this dependency. We integrated our CRISPR/Cas9-based data on MM-selective essential genes with a reanalysis of the Achilles Heel shRNA screen in MM and non-MM cell lines (10 and 493, respectively) of the Cell Line Encyclopedia Program (CCLE) program. We applied a series of statistical tests (e.g. Wilcoxon rank test or marker selection feature of GENE-E algorithm with 1000 permutation tests) to identify genes with a significantly lower rank in sgRNA or shRNA depletion in MM vs. non-MM cell lines, across different specific thresholds for fold change and statistical significance. We identified more than 50 high-value candidate target genes with preferential essentiality in MM, compared to non-MM cell lines of diverse lineages. Prominent examples of such MM-selective, essential genes included: transcription factors (e.g. IRF4, CCND2, MAF, NFKB1, NFKB2, RELA, RELB); otherNF-kB-related genes (e.g. IKBKB); PIM2 (but not PIM1 or PIM3 in this cell line panel); regulators of protein homeostasis, including diverse E2 and E3 ubiquitin ligases; and several other known or biologically-plausible dependencies which are under further evaluation. Many of these MM-selective dependencies exhibited significantly higher expression in MM, compared to non-MM cells, both in cell lines (based on the CCLE dataset) and patient-derived samples (comparison of Broad/MMRF vs. TCGA datasets, respectively). Notable observations of context-dependent essential genes include ARID1A in MM.1S cells (plausibly due to deficiency in its paralog ARID1B); and cases of both MM and non-MM cells with RAS mutations but lack of dependency on that gene. Targeting of lineage-specific dependencies (e.g. ER or AR in breast or prostate Ca, respectively) has provided major clinical benefit in some tumors; while context-specific dependencies are a cornerstone of molecularly-guided individualized treatments. Therefore, by identifying lineage- and context-dependent essential genes for MM, our integrated genome-wide CRISPR/Cas9 and shRNA analyses in molecularly annotated panels of MM vs. non-MM cell lines provide an attractive framework towards designing novel therapies for MM. DisclosuresNo relevant conflicts of interest to declare.

Abstract B086: CRISPR-Cas9 engineering and screening in primary human T cells

Abstract Recent clinical data indicate immunotherapy can be an effective treatment for cancer patients, yielding dramatically increased survival times in some cases. As not all patients benefit from treatments such as anti-CTLA4 and anti-PD1 antibodies, a major focus in immuno-oncology research is finding new immuno-oncology targets, including those that alter the character and frequency of T-cell-mediated anti-tumor responses. We have had great success using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas9 mediated genome editing to probe gene function in cancer cells via the generation of knock-out and knock-in mutants. We are now applying this approach to primary immune cells and are deploying both CRISPR–Cas9 cell engineering and CRISPR–Cas9 screens to better understand T cell biology and to find new therapeutic targets. We use T cells negatively purified by magnetic sorting from peripheral blood mononuclear cells taken from healthy donors. Our initial work in primary human T cells has focused on the capacity to knock out and knock in genes using the Neon™ transfection system. Our data indicate that primary T cells are amenable to gene editing, and the capacity to rapidly modify loci, such as PDCD1, which encodes PD-1 enables generation of primary T cell models suitable for comprehending the function of modified receptor–ligand pairs involved in an immune checkpoint response. Our pooled sgRNA–Cas9 screens have used our in-house sgRNA libraries, which include a modified tracrRNA component improving Cas9 affinity and subsequently the performance of a typical sgRNA for promoting gene editing and modifying phenotype. Briefly, isolated CD3+ T cells are stimulated in vitro with anti-CD3 and anti-CD28 antibodies in the presence of recombinant IL-2. Next, the proliferating T cells are co-cultured with a GFP expressing Lentivirus that directs expression of Cas9 and an sgRNA drawn from a 3900 member sgRNA library targeting genes involved in the regulation of metabolism and a control library of 2442 guide RNAs. After prolonged Lentivirus and T cell co-culture, transduced T cells are sorted based on their GFP expression and periodically re-stimulated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 to allow their proliferation and expansion over several weeks. Representative cell pellets are taken after GFP sorting and then at specific time points throughout the screen. For each time point, gDNA is extracted and PCR is carried out to isolate the gRNAs that are present in each cell and these are analysed using NGS. NGS results are interpreted using algorithms from the previously published model-based analysis of genome-wide CRISPR–Cas9 knockout (MAGeCK) and Bayesian normalisation of gene expression levels (BAGEL) approaches. These screens are currently ongoing and are being run in T cells isolated from five independent donors to assess the impact of donor variability. We will present results from these screens assessing guide drop-out kinetics and reproducibility by comparing the performance of specific guides over multiple time points in each of the donors. We anticipate that these data will be useful in building more complex screens that assess T cell biology in the presence of additional cells, such as myeloid derived suppressor cells, involved in regulating the immune response to tumor development and progression. Citation Format: Cristina Ghirelli, Thibault Laurent, Kim Hoenderdos, Chris Lowe, Nicola McCarthy, Jonathan Moore. CRISPR-Cas9 engineering and screening in primary human T cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B086.

CRISPR/Cas9 Technique for Identification of Genes Regulating Oxaliplatin Resistance of Pancreatic Cancer Cell Line

Genome editing approach based on prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) system is a simple and useful way to investigate gene functions on a genome-wide scale. It is especially important for cancer research because of genetic contribution to tumor development. We applied this technique in a high-throughput screening format to find genes that could be involved in chemotherapy resistance of pancreatic cancer. We used AsPC1 cell line expressing doxycycline-inducible Cas9 to screen two sgRNA lentiviral libraries: (1) cell cycle genes (CC, 983 genes, ∼12,000 sgRNA) and (2) genome-wide (GW, ∼90,000 sgRNA). These sets of cells with different gene knockouts were treated with oxaliplatin to identify knockouts which increase sensitivity to the drug. We have performed screening both in vitro and in vivo settings. For the in vivo arm of our experiments, peritoneal carcinomatosis model in severe combined immunodeficiency (SCID) mice was created by intraperitoneal injection of AsPC1/Cas9 cells infected with sgRNA library. Genomic DNA from cells and animal tumor material was analyzed using next generation sequencing (NGS) to obtain data about representation of sgRNA. Preliminary data allowed us to identify genes potentially modulating oxaliplatin sensitivity.

Identification of oncogenic driver mutations by genome-wide CRISPR-Cas9 dropout screening.

BackgroundGenome-wide CRISPR-Cas9 dropout screens can identify genes whose knockout affects cell viability. Recent CRISPR screens detected thousands of essential genes required for cellular survival and key cellular processes; however discovering novel lineage-specific genetic dependencies from the many hits still remains a challenge.ResultsTo assess whether CRISPR-Cas9 dropout screens can help identify cancer dependencies, we screened two human cancer cell lines carrying known and distinct oncogenic mutations using a genome-wide sgRNA library. We found that the gRNA targeting the driver mutation EGFR was one of the highest-ranking candidates in the EGFR-mutant HCC-827 lung adenocarcinoma cell line. Likewise, sgRNAs for NRAS and MAP2K1 (MEK1), a downstream kinase of mutant NRAS, were identified among the top hits in the NRAS-mutant neuroblastoma cell line CHP-212. Depletion of these genes targeted by the sgRNAs strongly correlated with the sensitivity to specific kinase inhibitors of the EGFR or RAS pathway in cell viability assays. In addition, we describe other dependencies such as TBK1 in HCC-827 cells and TRIB2 in CHP-212 cells which merit further investigation.ConclusionsWe show that genome-wide CRISPR dropout screens are suitable for the identification of oncogenic drivers and other essential genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3042-2) contains supplementary material, which is available to authorized users.

Abstract 876: Genome-scale genetic knockout screen identifies modifiers of EGFR dependence in non-small cell lung cancer cells

Abstract Non-small cell lung cancers with epidermal growth factor receptor (EGFR) gene mutations can exhibit a strong dependence on its signaling for growth and survival. They are also sensitive to EGFR tyrosine kinase inhibitors (TKIs), which provide superior clinical benefits to conventional chemotherapy. However, despite initial response, most patients experience relapse with resistant tumors within a year. This study aims to identify modifiers of dependence on mutant EGFR signaling and the mechanisms by which they do so in order to improve therapeutic strategies and outcomes. We first performed a genome-scale CRISPR-Cas9 genetic knockout screen. In this system, gene silencing occurs by site-specific double-strand-breaks induced by the Cas9 nuclease that is localized to its target by a single-guide RNA (sgRNA). The error-prone repair pathway introduces insertion/deletion mutations leading to frameshifts or premature stop codons. We constructed a pooled sgRNA library targeting more than 18,000 protein-coding human genes with multiple sgRNA vectors. The cell line HCC827 was used in our screen as it is EGFR-mutant and sensitive to EGFR TKIs. We transduced HCC827 lung cancer cells with the pooled sgRNA library and cultured them in the presence of Erlotinib, an EGFR TKI, or DMSO control for 17 days. At the end of the treatment period we identified sgRNAs that were enriched in Erlotinib-treated groups over control groups, indicating genes whose loss-of-function confer TKI resistance. We applied the RNAi gene enrichment ranking (RIGER) algorithm to identify gene hits with enrichment of multiple sgRNAs. Top-ranked candidates include previously confirmed genes PTEN, NF1, NF2, TSC1, and TSC2, validating this system as a means to identify modifiers of EGFR dependence in HCC827 cells. A novel candidate gene is the E3 ubiquitin ligase HUWE1. We showed that suppression of HUWE1 by sgRNAs or inducible short hairpin RNA (shRNA) in HCC827 cells re-activated AKT and ERK1/2 signaling pathways and increased cell proliferation and survival in response to EGFR inhibition. These findings were confirmed in vivo by implanting mouse xenografts of HCC827 cells expressing shHUWE1 and monitoring tumor development in response to EGFR-TKI therapy. Tumors expressing HUWE1 led to durable tumor regression throughout the treatment period but tumors with suppressed HUWE1 continued to grow into large tumors. Immunohistochemistry analysis of excised tumors also revealed increased AKT and ERK1/2 signaling as well as increased proliferation and decreased apoptosis. We have shown that dependence on EGFR signaling can be decreased in EGFR-mutant lung cancer cells through mechanisms that involve the activation of AKT and ERK1/2 signaling pathways. Ongoing studies involve identifying HUWE1 substrates/interactions that participate in tumor cell response to EGFR inhibitors, revealing a novel mechanism of resistance to EGFR-targeted therapy. Citation Format: Jon DiMaina, Chris Duckworth, Hiu Wing Cheung. Genome-scale genetic knockout screen identifies modifiers of EGFR dependence in non-small cell lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 876.

Abstract 2743: Finding determinants of PARP inhibitor sensitivity using genome-wide and focused CRISPR screens

Abstract We are using genomic approaches to study the mechanism of action of targeted cancer drugs, particularly poly(ADP-ribose) polymerase (PARP) inhibitors. One of these drugs, olaparib was recently approved for use in ovarian cancers with germline BRCA1/2 mutations and many other clinical trials with this and other PARP inhibitors are underway. Experimental study of potential resistance mechanisms can inform the ongoing clinical development of these drugs. We previously carried out a loss of function mutagenesis screen in haploid cells, which showed that PARP1 was required for cellular toxicity of olaparib and other PARP inhibitors. This supported the hypothesis that PARP inhibitor toxicity occurs via a poisoning mechanism where inhibited PARP is tightly bound to DNA, forming a toxic lesion. Recently we carried out a similar screen using genome-wide lentiviral CRISPR-Cas9 mutagenesis in diploid cells. Many PARP inhibitor resistant clones had loss of function Parp1 mutations as expected. However, we also isolated a point mutation affecting a single amino acid in the Parp1 DNA binding domain. This mutant encodes a stable Parp1 protein that cannot bind DNA and does not become trapped in the presence of inhibitors. Thus the CRISPR screen implicated PARP1 DNA binding directly in determination of PARP inhibitor toxicity and was extremely informative about the mechanism of action compared to conventional loss-of-function mutagenesis. We extended this approach by synthesising a high-density focused sgRNA library targeting only PARP1. We developed a reporter cell line that allows us to selectively isolate in-frame mutations that preserve PARP1 protein expression. By deep sequencing mutagenised and appropriately selected cells we identified a series of subtle mutations in PARP1 that result in PARP inhibitor resistance, giving us a detailed insight into structure-function relationships in PARP1. The approach is applicable to other drug targets, providing a framework for understanding drug action and predicting clinical resistance. Citation Format: Stephen Pettitt, Dragomir B. Krastev, Feifei Song, Alan Ashworth, Christopher J. Lord. Finding determinants of PARP inhibitor sensitivity using genome-wide and focused CRISPR screens. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2743.

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents.

TRUE gene silencing (termed after tRNase Z(L)-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3' processing endoribonuclease, tRNase Z(L), to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.