CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on-and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens.

Identification of effective combination therapies is critical to address the emergence of drug-resistant cancers, but direct screening of all possible drug combinations is infeasible. Here we introduce a CRISPR-based double knockout (CDKO) system that improves the efficiency of combinatorial genetic screening using an effective strategy for cloning and sequencing paired single-guide RNA libraries and a robust statistical scoring method for calculating genetic interactions (GIs) from CRISPR-deleted gene pairs. We applied CDKO to generate a large-scale human GI map, comprising 490,000 double-sgRNAs directed against 21,321 pairs of drug targets in K562 leukemia cells and identified synthetic lethal drug target pairs for which corresponding drugs exhibit synergistic killing. These included the BCL2L1 and MCL1 combination, which was also effective in imatinib-resistant cells. We further validated this system by identifying known and previously unidentified GIs between modifiers of ricin toxicity. This work provides an effective strategy to screen synergistic drug combinations at high-throughput and a CRISPR-based tool to dissect functional GI networks.

Acetylation of histone H3 at lysine 27 is a well-defined marker of enhancer activity. However, the functional impact of this modification at enhancers is poorly understood. Here, we use a chemical genetics approach to acutely block the function of the cAMP response element binding protein (CREB) binding protein (CBP)/P300 bromodomain in models of hematological malignancies and describe a consequent loss of H3K27Ac specifically from enhancers, despite the continued presence of CBP/P300 at chromatin. Using this approach to dissect the role of H3K27Ac at enhancers, we identify a critical role for this modification in the production of enhancer RNAs and transcription of enhancer-regulated gene networks.

Highlights d CRISPR screen reveals host genes regulating distinct steps of L. pneumophila infection d Previously uncharacterized genes C1ORF43 and KIAA1109 regulate phagocytosis d Host Rab10 is hijacked by SidC/SdcA to promote ER recruitment and bacterial replication

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Activating mutations in RAS GTPases drive many cancers, but limited understanding of less-studied RAS interactors, and of the specifi c roles of different RAS interactor paralogs, continues to limit target discovery. We developed a multistage discovery and screening process to systematically identify genes conferring RAS-related susceptibilities in lung adenocarcinoma. Using affi nity purifi cation mass spectrometry, we generated a protein-protein interaction map of RAS interactors and pathway components containing hundreds of interactions. From this network, we constructed a CRISPR dual knockout library targeting 119 RAS-related genes that we screened for KRAS -dependent genetic interactions (GI). This approach identifi ed new RAS effectors, including the adhesion controller RADIL and the endocytosis regulator RIN1, and >250 synthetic lethal GIs, including a potent KRAS -dependent interaction between RAP1GDS1 and RHOA. Many GIs link specifi c paralogs within and between gene families. These fi ndings illustrate the power of multiomic approaches to uncover synthetic lethal combinations specifi c for hitherto untreatable cancer genotypes. SIGNIFICANCE:We establish a deep network of protein-protein and genetic interactions in the RAS pathway. Many interactions validated here demonstrate important specifi cities and redundancies among paralogous RAS regulators and effectors. By comparing synthetic lethal interactions across KRAS -dependent and KRAS -independent cell lines, we identify several new combination therapy targets for RAS-driven cancers.

Identification of effective combination therapies is critical to address the emergence of drug-resistant cancers. Although millions of drug combinations might be created by repurposing existing drugs, direct screening of these combinations is infeasible. Here, we designed a scalable CRISPR-based double knockout (CDKO) system to generate a mammalian genetic interaction (GI) map at unprecedented scale, comprised of 490,000 double-sgRNAs directed against 21,321 pairs of drug targets. We first developed an efficient strategy for cloning and sequencing the libraries, as well as a robust statistical scoring method for calculating GIs from CRISPR-deleted gene pairs. We then extensively validated this system by identifying known and novel genetic interactions in the ricin pathway, and compared the GIs to known protein-protein interactions (PPIs). Using this validated system, we searched for rare synthetic lethal drug target pairs in K562 leukemia cells and identified a number of potent combinations for which corresponding drugs exhibit synergistic killing. Together, this work demonstrates an effective strategy to screen synergistic drug combinations in high throughput, and a powerful CRISPR-based tool to dissect functional genetic interaction networks. Citation Format: Kyuho Han, Edwin Jeng, Gaelen Hess, David Morgens, Amy Li, Michael Bassik. A CRISPR-based genetic interaction map identifies synergistic drug combinations for cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR04.

Dasatinib is a multi-tyrosine kinase inhibitor approved for treatment of Ph+ acute lymphoblastic leukemia (ALL), but its efficacy is limited by resistance. Recent preclinical studies suggest that dasatinib may be a candidate therapy in additional ALL subtypes including pre-BCR+ ALL. Here we utilized shRNA library screening and global transcriptomic analysis to identify several novel genes and pathways that may enhance dasatinib efficacy or mitigate potential resistance in human pre-BCR+ ALL. Depletion of the transcriptional co-activator CBP increased dasatinib sensitivity by downregulating transcription of the pre-BCR signaling pathway previously associated with dasatinib sensitivity. Acquired resistance was due in part to upregulation of alternative pathways including WNT through a mechanism suggesting transcriptional plasticity. Small molecules that disrupt CBP interactions with the CREB KID domain or β-catenin showed promising preclinical efficacy in combination with dasatinib. These findings highlight novel modulators of sensitivity to targeted therapies in human pre-BCR+ ALL, which can be reversed by small molecules inhibitors. They also identify promising therapeutic approaches to ameliorate dasatinib sensitivity and prevent resistance in ALL.