Guide RNA Library Research Articles

Minimal genome-wide human CRISPR-Cas9 library

CRISPR guide RNA libraries have been iteratively improved to provide increasingly efficient reagents, although their large size is a barrier for many applications. We design an optimised minimal genome-wide human CRISPR-Cas9 library (MinLibCas9) by mining existing large-scale gene loss-of-function datasets, resulting in a greater than 42% reduction in size compared to other CRISPR-Cas9 libraries while preserving assay sensitivity and specificity. MinLibCas9 provides backward compatibility with existing datasets, increases the dynamic range of CRISPR-Cas9 screens and extends their application to complex models and assays.

DNA polymerase ι compensates for Fanconi anemia pathway deficiency by countering DNA replication stress

Fanconi anemia (FA) is caused by defects in cellular responses to DNA crosslinking damage and replication stress. Given the constant occurrence of endogenous DNA damage and replication fork stress, it is unclear why complete deletion of FA genes does not have a major impact on cell proliferation and germ-line FA patients are able to progress through development well into their adulthood. To identify potential cellular mechanisms that compensate for the FA deficiency, we performed dropout screens in FA mutant cells with a whole genome guide RNA library. This uncovered a comprehensive genome-wide profile of FA pathway synthetic lethality, including POLI and CDK4 As little is known of the cellular function of DNA polymerase iota (Pol ι), we focused on its role in the loss-of-function FA knockout mutants. Loss of both FA pathway function and Pol ι leads to synthetic defects in cell proliferation and cell survival, and an increase in DNA damage accumulation. Furthermore, FA-deficient cells depend on the function of Pol ι to resume replication upon replication fork stalling. Our results reveal a critical role for Pol ι in DNA repair and replication fork restart and suggest Pol ι as a target for therapeutic intervention in malignancies carrying an FA gene mutation.

Abstract 2154: Immune pressure selects ANKRD52 mutations for cancer cells to escape T cell-mediated killing

Abstract Cancer arises after somatic mutations override cellular and immunological control of cell proliferation. Mutations in oncogenes and tumor suppressor genes endow cancer cells intrinsic growth advantage over neighboring cells. Mutations can also enable cancer cells to survive immune clearance during cancer progression, but systematic analysis of such mutations is lacking. We hypothesized that some genetic alterations carried in cancer cell lines might specifically affect the cell fitness to T cell-mediated killing. By deep sequencing of syngeneic xenograft tumors receiving different levels of immune pressure, we enriched 53 mutations in clonal populations against T cell immunity. To differentiate the driver from passenger mutations, we performed in vivo and T cell co-culture CRISPR screens with a guide RNA library targeting these candidate genes. We identified ANKRD52, a promoter of miRNA-mediated silencing, as required for PD-1-independent T cell cytotoxicity by activating the JAK-STAT signaling and antigen presentation in cancer cells. ANKRD52 mutations found in prevalent cancer patients failed to activate the interferon-γ pathway and these patients exhibit poor overall survival. We hereby provide an approach for interrogating cancer genetic heterogeneity to uncover evolutionarily conserved evasion mechanism from host immunity. Citation Format: Tianyu Y. Song, Haixin Zhao, Hongjie Fan, Min Long, Chenlu Geng, Xiaoxiao Xie, Yang Liu, Wenrong Zhou, Ziyu Chen, Dawei Huang, Bo Peng, Zhengang Peng, Yong Cang. Immune pressure selects ANKRD52 mutations for cancer cells to escape T cell-mediated killing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2154.

A Three-Way Combinatorial CRISPR Screen for Analyzing Interactions among Druggable Targets

We present a CRISPR-based multi-gene knockout screening system and toolkits for extensible assembly of barcoded high-order combinatorial guide RNA libraries en masse. We apply this system for systematically identifying not only pairwise but also three-way synergistic therapeutic target combinations and successfully validate double- and triple-combination regimens for suppression of cancer cell growth and protection against Parkinson's disease-associated toxicity. This system overcomes the practical challenges of experimenting on a large number of high-order genetic and drug combinations and can be applied to uncover the rare synergistic interactions between druggable targets.

An Isothermal Method for Sensitive Detection of Mycobacterium tuberculosis Complex Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a Cis and Trans Cleavage

Tuberculosis is one of the most serious infectious diseases, resulting in death worldwide. Traditional detection methods are not enough to meet the clinical requirements of rapid diagnosis, high specificity, and high sensitivity. Fast, sensitive, and accurate detection of Mycobacterium tuberculosis (MTB) is urgently needed to treat and control tuberculosis disease. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas12a) exhibit strong nonspecific degradation ability of exogenous single-strand nucleic acids (trans cleavage) after specific recognition of target sequence. We purified Cas12a protein and selected a proper guide RNA based on conserved sequences of MTB from designed guide RNA library. Then, we proposed a novel detection method based on recombinase polymerase amplification and CRISPR/Cas12a nuclease system for specific and sensitive detection of MTB DNA. The assay, based on fluorescence detection, showed 4.48 fmol/L of limit of detection and good linear correlation of concentration with fluorescence value (R2=0.9775). It also showed good performance in distinguishing other bacteria. Furthermore, its clinical performance was evaluated by 193 samples and showed sensitivity of 99.29% (139/140) and specificity of 100% (53/53) at 99% CI, compared with culture method. Taken together, the CRISPR/Cas12a system showed good specificity, excellent sensitivity, and excellent accuracy for MTB detection, and it meets requirements of MTB detection in clinical samples and has great potential for clinical translation.

Parallel CRISPR-Cas9 screens clarify impacts of p53 on screen performance.

CRISPR-Cas9 genome engineering has revolutionised high-throughput functional genomic screens. However, recent work has raised concerns regarding the performance of CRISPR-Cas9 screens using TP53 wild-type human cells due to a p53-mediated DNA damage response (DDR) limiting the efficiency of generating viable edited cells. To directly assess the impact of cellular p53 status on CRISPR-Cas9 screen performance, we carried out parallel CRISPR-Cas9 screens in wild-type and TP53 knockout human retinal pigment epithelial cells using a focused dual guide RNA library targeting 852 DDR-associated genes. Our work demonstrates that although functional p53 status negatively affects identification of significantly depleted genes, optimal screen design can nevertheless enable robust screen performance. Through analysis of our own and published screen data, we highlight key factors for successful screens in both wild-type and p53-deficient cells.

Delivering Robust Candidates to the Drug Pipeline through Computational Analysis of Arrayed CRISPR Screens.

Genome-wide arrayed CRISPR screening is a powerful method for drug target identification as it enables exploration of the effect of individual gene perturbations using diverse highly multiplexed functional and phenotypic assays. Using high-content imaging, we can measure changes in biomarker expression, intracellular localization, and cell morphology. Here we present the computational pipeline we have developed to support the analysis and interpretation of arrayed CRISPR screens. This includes evaluating the quality of guide RNA libraries, performing image analysis, evaluating assay results quality, data processing, hit identification, ranking, visualization, and biological interpretation.

Targeted CRISPR/Cas9-based in vitro and in vivo screening of metabolic pathways in primary T cells

Abstract Chronic inflammation and immunosuppression are characterized by a dysregulation in the balance between pro-inflammatory effector T (Teff) cells and anti-inflammatory regulatory T (Treg) cells. While many currently available therapies broadly target the immune compartment, selectively targeting the specific T cell subsets that contribute to disease may provide a new avenue for development of improved immunotherapies. We have previously shown that Teff and Treg cells can be distinguished by their reliance on distinct metabolic programs, and that this exposes a way to preferentially target specific T cell subsets. We have developed in vitro and in vivo approaches to CRISPR/Cas9-based genetic screening in primary T cells to interrogate metabolic pathways. In this method, we design small-scale targeted guide RNA libraries, which are transduced into primary T cells. For the in vitro assays, these cells can be cultured under selective pressures, such as with differentiation cytokines, and sorted for populations of interest. For the in vivo assays, the transduced cells are adoptively transferred into host mice of inflammatory or cancer models, such as airway inflammation or melanoma. We have constructed and tested a library targeting the solute carrier (SLC) family of transporters, which import and export essential nutrients required to fuel the metabolic programs. Preliminary results show the glutamine transporter, SLC38a2 (SNAT2), in addition to the previously identified transporter SLC7a5 (LAT1), to be critical for primary T cell proliferative capacity. This approach provides a powerful tool for identifying critical nodes within metabolic pathways of interest in primary T cells that can potentially serve as therapeutic targets.

On-target activity predictions enable improved CRISPR-dCas9 screens in bacteria.

The ability to block gene expression in bacteria with the catalytically inactive mutant of Cas9, known as dCas9, is quickly becoming a standard methodology to probe gene function, perform high-throughput screens, and engineer cells for desired purposes. Yet, we still lack a good understanding of the design rules that determine on-target activity for dCas9. Taking advantage of high-throughput screening data, we fit a model to predict the ability of dCas9 to block the RNA polymerase based on the target sequence, and validate its performance on independently generated datasets. We further design a novel genome wide guide RNA library for E. coli MG1655, EcoWG1, using our model to choose guides with high activity while avoiding guides which might be toxic or have off-target effects. A screen performed using the EcoWG1 library during growth in rich medium improved upon previously published screens, demonstrating that very good performances can be attained using only a small number of well designed guides. Being able to design effective, smaller libraries will help make CRISPRi screens even easier to perform and more cost-effective. Our model and materials are available to the community through crispr.pasteur.fr and Addgene.

In Vivo Epigenetic CRISPR Screen Identifies Asf1a as an Immunotherapeutic Target in Kras-Mutant Lung Adenocarcinoma.

Despite substantial progress in lung cancer immunotherapy, the overall response rate in patients with KRAS-mutant lung adenocarcinoma (LUAD) remains low. Combining standard immunotherapy with adjuvant approaches that enhance adaptive immune responses-such as epigenetic modulation of antitumor immunity-is therefore an attractive strategy. To identify epigenetic regulators of tumor immunity, we constructed an epigenetic-focused single guide RNA library and performed an in vivo CRISPR screen in a Kras G12D/Trp53 -/- LUAD model. Our data showed that loss of the histone chaperone Asf1a in tumor cells sensitizes tumors to anti-PD-1 treatment. Mechanistic studies revealed that tumor cell-intrinsic Asf1a deficiency induced immunogenic macrophage differentiation in the tumor microenvironment by upregulating GM-CSF expression and potentiated T-cell activation in combination with anti-PD-1. Our results provide a rationale for a novel combination therapy consisting of ASF1A inhibition and anti-PD-1 immunotherapy. SIGNIFICANCE: Using an in vivo epigenetic CRISPR screen, we identified Asf1a as a critical regulator of LUAD sensitivity to anti-PD-1 therapy. Asf1a deficiency synergized with anti-PD-1 immunotherapy by promoting M1-like macrophage polarization and T-cell activation. Thus, we provide a new immunotherapeutic strategy for this subtype of patients with LUAD.See related commentary by Menzel and Black, p. 179.This article is highlighted in the In This Issue feature, p. 161.

Synthetic Lethal Screen to Identify Molecular Mechanisms that Drive Uveal Melanoma

Melanoma that strikes the uveal cells of the eye represents about 5% of all melanoma cases. Uveal melanoma is a highly aggressive form of cancer that exhibits a propensity to metastasize to the liver. At this time there are no effective treatments for metastatic uveal melanoma and new therapeutic strategies are urgently needed. The majority of uveal melanomas are derived from early activating mutations in the G-proteins GNA11 or GNAQ. In order to further explore and identify molecular mechanisms that uveal melanoma cell lines rely upon for proliferation and survival, we will perform CRISPR/Cas9 mediated synthetic lethality screening. To accomplish this, we will generate a uveal melanoma cell line 92.1 that expresses the endonuclease Cas9 and introduce a guide RNA library into it that targets the functional domains of epigenetic regulator proteins. Thus, we will be able to determine which epigenetic regulator genes are required for the survival and proliferation of these cells by measuring which guide RNAs "drop out" over time. The guide RNAs that drop out likely disrupt essential genes that the cell relies on for growth. Cells carrying these gRNAs are consequently depleted from the culture. This will indicate that Cas9 mediated disruption of the gene targeted by that guide RNA causes cell death and will therefore identify the key dependencies of the uveal melanoma cells. We expect that the results of these experiments will reveal novel genetic mechanisms characteristic of GNAQ mutant uveal melanoma cells which could be therapeutically targeted in the future.

SliceIt: A genome-wide resource and visualization tool to design CRISPR/Cas9 screens for editing protein-RNA interaction sites in the human genome.

Several protein-RNA cross linking protocols have been established in recent years to delineate the molecular interaction of an RNA Binding Protein (RBP) and its target RNAs. However, functional dissection of the role of the RBP binding sites in modulating the post-transcriptional fate of the target RNA remains challenging. CRISPR/Cas9 genome editing system is being commonly employed to perturb both coding and noncoding regions in the genome. With the advancements in genome-scale CRISPR/Cas9 screens, it is now possible to not only perturb specific binding sites but also probe the global impact of protein-RNA interaction sites across cell types. Here, we present SliceIt (http://sliceit.soic.iupui.edu/), a database of in silico sgRNA (single guide RNA) library to facilitate conducting such high throughput screens. SliceIt comprises of ~4.8 million unique sgRNAs with an estimated range of 2-8 sgRNAs designed per RBP binding site, for eCLIP experiments of >100 RBPs in HepG2 and K562 cell lines from the ENCODE project. SliceIt provides a user friendly environment, developed using advanced search engine framework, Elasticsearch. It is available in both table and genome browser views facilitating the easy navigation of RBP binding sites, designed sgRNAs, exon expression levels across 53 human tissues along with prevalence of SNPs and GWAS hits on binding sites. Exon expression profiles enable examination of locus specific changes proximal to the binding sites. Users can also upload custom tracks of various file formats directly onto genome browser, to navigate additional genomic features in the genome and compare with other types of omics profiles. All the binding site-centric information is dynamically accessible via "search by gene", "search by coordinates" and "search by RBP" options and readily available to download. Validation of the sgRNA library in SliceIt was performed by selecting RBP binding sites in Lipt1 gene and designing sgRNAs. Effect of CRISPR/Cas9 perturbations on the selected binding sites in HepG2 cell line, was confirmed based on altered proximal exon expression levels using qPCR, further supporting the utility of the resource to design experiments for perturbing protein-RNA interaction networks. Thus, SliceIt provides a one-stop repertoire of guide RNA library to perturb RBP binding sites, along with several layers of functional information to design both low and high throughput CRISPR/Cas9 screens, for studying the phenotypes and diseases associated with RBP binding sites.

MultiGuideScan: a multi-processing tool for designing CRISPR guide RNA libraries.

The recent advance in genome engineering technologies based on CRISPR/Cas9 system is enabling people to systematically understand genomic functions. A short RNA string (the CRISPR guide RNA) can guide the Cas9 endonuclease to specific locations in complex genomes to cut DNA double-strands. The CRISPR guide RNA is essential for gene editing systems. Recently, the GuideScan software is developed to design CRISPR guide RNA libraries, which can be used for genome editing of coding and non-coding genomic regions effectively. However, GuideScan is a serial program and computationally expensive for designing CRISPR guide RNA libraries from large genomes. Here, we present an efficient guide RNA library designing tool (MultiGuideScan) by implementing multiple processes of GuideScan. MultiGuideScan speeds up the guide RNA library designing about 9-12 times on a 32-process mode comparing to GuideScan. MultiGuideScan makes it possible to design guide RNA libraries from large genomes. https://github.com/bioinfomaticsCSU/MultiGuideScan. Supplementary data are available at Bioinformatics online.

Abstract 2761: Development of synthetic guide RNA libraries for CRISPR-mediated transcriptional activation screening for gain-of-function studies

Abstract Functional gene analysis studies have been empowered by development of CRISPR-Cas9 gene knockout tools, however the CRISPR-Cas9 system has also been adapted for inhibition or activation of gene transcription. A nuclease-deactivated Cas9 (dCas9) can be fused to various effector domains to produce an RNA-guided transcription factor for either inhibition (CRISPRi) or activation (CRISPRa) of target genes. For overexpression studies, CRISPRa holds significant advantages over traditional vector-based gene expression, because genes are upregulated from their native promoter and endogenous genomic context. The majority of CRISPRa research performed to date has utilized single guide RNA (sgRNA) expressed from a DNA vector, primarily in the context of pooled lentiviral screening approaches. Here we describe the development of CRISPRa synthetic guide RNAs for the use in arrayed screening, so that we combine this next-generation transcriptional activation method with the ability to support more complex assays in a one-gene-per well format. Considerations for arrayed activation screens will be shown. The combination of gain-of-function from CRISPRa with loss-of-function using RNAi or canonical CRISPR-Cas9 for gene knockout allows for robust characterization of gene mechanisms and pathways. Citation Format: Annaleen Vermeulen, Zaklina Strezoska, Sarah M. Dickerson, Maren M. Gross, Eldon T. Chou, Elena Maksimova, Matthew R. Perkett, Shawn McClelland, Anja V. Smith. Development of synthetic guide RNA libraries for CRISPR-mediated transcriptional activation screening for gain-of-function studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2761.

S859 GENOME‐WIDE CRISPR‐CAS9 SCREEN REVEALS SYNTHETIC LETHALITY OF WNT PATHWAY ACTIVATION AND ASPARAGINASE IN DRUG‐RESISTANT ACUTE LEUKEMIAS

Background:Intensification of asparaginase‐based therapy has improved outcomes for several subtypes of acute leukemia, but the development of treatment resistance has a poor prognosis, and effective therapeutic options are lacking for many of these patients.Aims:We hypothesized, from the concept of synthetic lethality, that asparaginase‐resistant leukemia cells harbor gain‐of‐fitness alterations that could be exploited therapeutically.Methods:To identify molecular pathways that promote leukemic cell fitness upon treatment with asparaginase, we performed a genome‐wide CRISPR‐Cas9 loss‐of‐function genetic screen in the asparaginase‐resistant T‐ALL cell line CCRF‐CEM. Cas9‐expressing cells were transduced with the GeCKO genome‐wide guide RNA library, treated with either vehicle or low‐dose asparaginase, and guide RNA representation was assessed.Results:Our internal positive control, asparagine synthetase, was the gene most significantly depleted in asparaginase‐treated cells (RRA significance score = 1.56 x 10−7), followed closely by two regulators of Wnt signaling, NKD2 and LGR6 (RRA score = 6 x 10−6 and 2.19 x 10−5). Knockdown of NKD2 or LGR6 increased levels of active β‐catenin, as well as the activity of a TopFLASH reporter of canonical Wnt/β‐catenin transcriptional activity (P < 0.0001), indicating that both genes are negative regulators of Wnt signaling in these cells. Loss of NDK2 or LGR6 profoundly sensitized several acute leukemia cell lines to asparaginase (P < 0.0001), and potentiated asparaginase‐induced apoptosis (P < 0.0001). Thus, Wnt pathway activation sensitizes leukemic cells to asparaginase.Wnt signaling inhibits glycogen synthase kinase 3 (GSK3) activity, prompting us to test whether pharmacologic GSK3 inhibition can enhance asparaginase induced cytotoxicity. Treatment with CHIR99021 (an ATP‐competitive inhibitor of GSK3α and GSK3β) induced profound asparaginase sensitization in a panel of treatment‐resistant acute leukemia cell lines, including T‐ALL, MLL‐rearranged AML and hypodiploid B‐ALL (P < 0.0001), but not in normal hematopoietic progenitors.Sensitization to asparaginase was independent of β‐catenin and mTOR, but instead mediated by Wnt‐dependent stabilization of proteins (Wnt/STOP), which inhibits GSK3‐dependent protein ubiquitination and proteasomal degradation (Acebron et al. Mol Cell 2014). Proteasomal degradation is a catabolic source of amino acids, and we found that Wnt/STOP activation promoted leukemic cell death by exacerbating asparaginase‐induced depletion of asparagine (P = 0.017). Indeed, expression of a hyperactive PSMA4 proteasomal subunit that broadly increases proteasomal degradation (Choi et al. Nat Comm. 2016) completely blocked Wnt‐induced sensitization to asparaginase (P < 0.0001).To explore the therapeutic potential of our findings, we focused on GSK3 as a therapeutic target. Despite redundancy of GSK3α and GSK3β for many of their biologic functions, we found that depletion of GSK3α fully phenocopied Wnt‐induced asparaginase sensitization (P < 0.0001), whereas GSK3β inhibition had no effect. Thus, we tested the combination of the GSK3α selective inhibitor BRD0705 (Wagner et al. Sci Transl Med 2018) and asparaginase in NRG mice, which were injected with patient‐derived xenografts (PDXs) from different acute leukemia subtypes. All PDXs proved completely refractory to asparaginase monotherapy, but the combination was highly efficacious (Fig.1, P < 0.0001).Summary/Conclusion:These results provide a compelling rationale for clinical trials testing asparaginase in combination with GSK3α inhibitors for the treatment of acute leukemias.image

Beditor: A Computational Workflow for Designing Libraries of Guide RNAs for CRISPR-Mediated Base Editing.

CRISPR-mediated base editors have opened unique avenues for scar-free genome-wide mutagenesis. Here, we describe a comprehensive computational workflow called beditor that can be broadly adapted for designing guide RNA libraries with a range of CRISPR-mediated base editors, Protospacer Adjacent Motif (PAM) recognition sequences, and genomes of many species. Additionally, to assist users in selecting the best sets of guide RNAs for their experiments, a priori estimates of editing efficiency, called beditor scores, are calculated. These beditor scores are intended to select guide RNAs that conform to requirements for optimal base editing: the editable base falls within maximum activity window of the CRISPR-mediated base editor and produces nonconfounding mutational effects with minimal predicted off-target effects. We demonstrate the utility of the software by designing guide RNAs for base editing to model or correct thousands of clinically important human disease mutations.

Abstract A065: Genome-scale CRISPR screens identify essential genes for tumor sensitivity to NK cells

Abstract Harnessing natural killer (NK) cells to attack tumors could improve immune-based cancer treatment strategies. However, mechanisms regulating sensitivity or resistance of cancer cells to the effector function of NK cells are incompletely understood. Here, we performed genome-scale CRISPR-Cas9 loss-of-function screens in human cancer cells to discover genes that influence susceptibility to primary human NK cells. To screen for genes essential for the interaction between NK cells and cancer cells, we infected human cancer cells expressing Cas9 with a genome-scale lentiviral guide RNA library. The resulting pool of knockout cells was exposed to NK cells expanded from peripheral blood of healthy donors. Enriched and depleted knockout clones were detected by next-generation sequencing of the integrated sgRNA cassettes, enabling identification of genes conferring resistance or susceptibility to NK cell-mediated lysis. The screens were performed in cell lines from diverse cancer types, including chronic myeloid leukemia (CML), B cell acute lymphoblastic leukemia, diffuse large B-cell lymphoma (DLBCL), and multiple myeloma. We recovered several known mechanisms of NK cell/cancer cell interactions, demonstrating feasibility of the screening approach. Loss of genes encoding components of the MHC class I complex (B2M, HLA-A, HLA-C, HLA-E) sensitized multiple cancer cell lines to NK cell-mediated lysis. This is consistent with missing-self recognition as a fundamental mechanism of NK cell activation. Furthermore, knockout of IFN-JAK-STAT signaling mediators led to increased tumor cell lysis, suggesting that MHC class I induction in response to NK cell-derived IFN gamma enables NK cell evasion by tumor cells. We also identified genes essential for effective NK cell-mediated lysis. NCR3LG1, encoding the B7-H6 ligand for the NKp30 activating NK cell receptor, was essential for NK cell lysis of CML cells. In contrast, knockout of apoptotic mediators and TRAIL pathway components conferred resistance to NK cell cytotoxicity in DLBCL cells, indicating heterogeneity in NK cell/cancer cell interactions between cancer types. Our data support a view that distinct mechanisms regulate sensitivity to NK cell cytotoxicity in different cancers. Importantly, our results indicate that loss-of-function mutations in the antigen-presenting machinery and the IFN-JAK-STAT pathway sensitize tumors to NK cell effector function. As alterations in these genes are associated with resistance to T-cell immunotherapies such as PD-1 blockade, NK cell-based therapies could be employed to overcome resistance in these patients. In summary, we suggest that systematic identification of mechanisms governing tumor immune susceptibility has the potential to uncover novel immunotherapy targets. Citation Format: Olli Dufva, Jay Klievink, Khalid Saeed, Matti Kankainen, Mette Ilander, Tiiina Hannunen, Sonja Lagström, Pekka Ellonen, Dean A Lee, Satu Mustjoki. Genome-scale CRISPR screens identify essential genes for tumor sensitivity to NK cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A065.

JACKS: joint analysis of CRISPR/Cas9 knockout screens.

Genome-wide CRISPR/Cas9 knockout screens are revolutionizing mammalian functional genomics. However, their range of applications remains limited by signal variability from different guide RNAs that target the same gene, which confounds gene effect estimation and dictates large experiment sizes. To address this problem, we report JACKS, a Bayesian method that jointly analyzes screens performed with the same guide RNA library. Modeling the variable guide efficacies greatly improves hit identification over processing a single screen at a time and outperforms existing methods. This more efficient analysis gives additional hits and allows designing libraries with a 2.5-fold reduction in required cell numbers without sacrificing performance compared to current analysis standards.

Comprehensive Integrated Genomic Perturbations Reveal Molecular Mechanisms of Red Blood Cell Trait Associations

Discovery of molecular mechanisms responsible for trait associations as discovered by genome-wide association studies (GWAS) is hampered by difficulty in identifying causal genetic variants due to linkage disequilibrium. Typical assays of genetic function are low throughput or evaluate sequences in heterologous ectopic settings. Genome editing enables perturbation of trait-associated genetic sequences within relevant genomic, chromatin and cellular context. Here we perform comprehensive analysis of genetic variants associated with red blood cell traits by pooled CRISPR screening. We performed a genome-wide Cas9 gene knockout screen in immortalized erythroid precursors (HUDEP-2 cells) during erythroid maturation to define functional erythroid genes required for cell growth or differentiation. We evaluated 952 loci associated with nine red blood cell traits (Astle et al, Cell 2016) comprising 24,843 SNPs. We linked 7,187 (28.9%) of these SNPs to genes by at least one of four routes: sharing topological associated domain, physical proximity (<20 kb), long-range chromatin interaction (promoter HiC), or eQTL with a functional erythroid gene. We designed ~5 guide RNAs per SNP requiring cleavage position within at least 50 bp and exceeding an off-target score threshold, resulting in 32,710 sgRNAs testing 5,592 SNPs at 481 loci. We utilized four editors: Cas9 nuclease to produce indels, dCas9-VP64 for gene activation, dCas9-KRAB for gene repression, and dCas9 as a DNA targeting control. By pooled lentiviral transduction, erythroid differentiation culture, and guide RNA library deep sequencing, we found reproducible results across biological replicates, with guide count clustered by Cas9 protein type. We performed fine-mapping of association results by Bayesian inference to calculate posterior probability of inclusion (PPI). We found a strong correlation between PPI and CRISPR significance score indicating agreement between CRISPR screening and genetic fine-mapping, despite examples of validated CRISPR signals with low PPI scores. We identified numerous CRISPR-implicated functional SNPs at regulatory elements including promoters and enhancers but also at noncoding sequences lacking chromatin marks. The editing of CRISPR -implicated functional SNPs correlated well with expression of linked genes. Editing of CRISPR-implicated functional SNPs caused altered proliferation and/or differentiation of both HUDEP-2 cells and CD34+ HSPC-derived primary erythroid precursors. We validated a functional SNP at the BCL2L1 enhancer, where both Cas9 disruption and dCas9-KRAB inhibition resulted in decreased expression of BCL2L1,and reduced cell survival during erythropoiesis. We validated several CRISPR-implicated functional SNPs at GFI1B that controlled erythroid cell differentiation, including one at a distal enhancer element and another that impacted exon splicing. These results demonstrate the potential and challenges of comprehensive integrated genomic perturbation to complement genetic, biochemical, and statistical approaches to uncover molecular underpinnings of human blood cell traits. DisclosuresNo relevant conflicts of interest to declare.

Synthetic Lethality of Wnt Pathway Activation and Asparaginase in Drug-Resistant Acute Leukemias

Asparaginase, a bacterial enzyme that depletes the nonessential amino acid asparagine, is an integral component of acute leukemia therapy. However, asparaginase resistance is a common clinical problem whose biologic basis is poorly understood. We hypothesized, based on the concept of synthetic lethality, that gain-of-fitness alterations in the drug-resistant cells had conferred a survival advantage that could be exploited therapeutically.To identify molecular pathways that promote fitness of leukemic cells upon treatment with asparaginase, we performed a genome-wide CRISPR/Cas9 loss-of-function screen in the asparaginase-resistant T-ALL cell line CCRF-CEM. Cas9-expressing CCRF-CEM cells were transduced with a genome-wide guide RNA library (Shalem et al. Science343, 84-87, 2014), treated with either vehicle or asparaginase (10 U/L), and guide RNA representation was assessed. Our internal positive control, asparagine synthetase, was the gene most significantly depleted in asparaginase-treated cells (RRA significance score = 1.56 x 10-7), followed closely by two regulators of Wnt signaling, NKD2 and LGR6 (RRA score = 6 x 10-6and 2.19 x 10-5, respectively). To test how these genes regulate Wnt signaling in T-ALL, we transduced CCRF-CEM cells with shRNAs targeting NKD2 or LGR6, or with an shLuciferase control. Knockdown of NKD2 or LGR6 increased levels of active β-catenin, as well as the activity of a TopFLASH reporter of canonical Wnt/β-catenin transcriptional activity (P < 0.0001), indicating that NKD2 and LGR6 are negative regulators of Wnt signaling in these cells. We then validated the screen results using shRNA knockdown of NKD2 or LGR6, which profoundly sensitized these cells to asparaginase (P< 0.0001) and potentiated asparaginase-induced apoptosis (P < 0.0001).Inhibition of glycogen synthase kinase 3 (GSK3) is a key event in Wnt-induced signal transduction. Thus, we tested whether CHIR99021, an ATP-competitive inhibitor of both GSK3 isoforms (GSK3α and GSK3β), could phenocopy the effect of Wnt pathway activation. Pharmacologic inhibition of GSK3 induced significant sensitization to asparaginase across a panel of cell lines representing distinct subtypes of treatment-resistant acute leukemia, including T-ALL, AML and hypodiploid B-ALL (Fig. 1a, b). Importantly, GSK3 inhibition did not sensitize normal hematopoietic progenitors to asparaginase, suggesting a leukemia-specific effect.Wnt-induced sensitization to asparaginase was independent of β-catenin and mTOR activation, because genetic and pharmacologic manipulation of these Wnt targets had no effect on asparaginase response. Instead, it was mediated by Wnt-dependent stabilization of proteins (Wnt/STOP), which inhibits GSK3-dependent protein ubiquitination and proteasomal degradation (Acebron et al. Mol Cell54, 663-674, 2014, Taelman et al. Cell143, 1136-1148, 2010). Indeed,Wnt-induced sensitization to asparaginase was completely blocked by the transduction of leukemia cells with FBXW7 (P < 0.0001), whose overexpression can reverse Wnt/STOP (Acebron et al. Mol Cell54, 663-674; 2014), or by expression of a hyperactive proteasomal subunit ΔN-PSMA4 (P < 0.0001), which globally increases protein degradation (Choi et al. Nat Commun7, 10963, 2016).Although GSK3α and GSK3βare redundant for many of their biologic functions, genetic or pharmacologic inhibition of GSK3α fully phenocopied Wnt-induced sensitization to asparaginase (P < 0.0001), whereas selective inhibition of GSK3β had no effect. We then leveraged the recently developed GSK3α-selective small molecule inhibitor BRD0705 (Wagner et al. Sci Transl Med10, 2018) to test the in vivo therapeutic potential of our findings. Immunodeficient NRG mice were injected with leukemic cells from a primary asparaginase-resistant T-ALL patient derived xenograft, and treatment was begun after confirmation of leukemic engraftment (n=16 mice per group). In vivo, this PDX proved completely resistant to asparaginase or BRD0705 monotherapy, whereas the combination was highly efficacious (median survival of 17 days in vehicle, vs. median not reached at 60 days in combo-treated mice; P < 0.0001; Fig. 2a, b). The combination was also well-tolerated, with no appreciable weight changes or increases in serum bilirubin levels.Our findings provide a molecular rationale for activating Wnt/STOP signaling to improve the therapeutic index of asparaginase. DisclosuresNo relevant conflicts of interest to declare.