Project description:We present a CRISPR-based multi-gene knockout screening system and new 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 validated double and triple combination regimens for suppression on cancer cell growth and protection against Parkinson’s disease-associated toxicity. This system overcomes the practical challenges to experiment on a large number of high-order genetic and drug combinations, and is applicable for uncovering the rare synergistic interactions between druggable targets.
Project description:Chromatin regulators are frequently mutated in human cancer and are attractive drug targets. They include diverse proteins that share functional domains and assemble into related multi-subunit complexes. To investigate functional relationships among these regulators, here we apply combinatorial CRISPR knockouts (KOs) to test over 35,000 gene-gene pairings in leukemia cells, using a library of over 300,000 constructs. Top pairs that demonstrate either compensatory non-lethal interactions or synergistic lethality enrich for paralogs and targets that occupy the same protein complex. The screen highlights protein complex dependencies not apparent in single KO screens, for example MCM histone exchange, the nucleosome remodeling and deacetylase (NuRD) complex, and HBO1 (KAT7) complex. We explore two approaches to NuRD complex inactivation. Paralog and non-paralog combinations of the KAT7 complex emerge as synergistic lethal and specifically nominate the ING5 PHD domain as a potential therapeutic target when paired with other KAT7 complex member losses. These findings highlight the power of combinatorial screening to provide mechanistic insight and identify therapeutic targets within redundant networks.
Project description:Docetaxel chemotherapy in metastatic prostate cancer offers only a modest survival benefit due to emerging resistance. To identify candidate therapeutic gene targets, we applied a murine prostate cancer orthograft model that recapitulates clinical invasive prostate cancer in a genome-wide CRISPR/Cas9 screen under docetaxel treatment pressure.
Project description:Combinatorial genetic perturbations have been utilized to identify synthetic sick/lethal genetic interactions for cancer drug target discovery. Current methods for high-throughput combinatorial genetic screening are inefficient and cumbersome. Here we developed a simple, robust, and high-performance CRISPR-Cas12a-based approach for unbiased, combinatorial genetic screening in cancer cells.
Project description:A number of reports have demonstrated that tumor-intrinsic mechanisms of resistance, such as the loss of genes critical for antigen presentation and inflammatory responses, along with the activation of various cellular signaling cascades, can limit the efficacy of immunotherapy. Strategies to sensitize tumor cells to immunotherapy may overcome some resistance mechanisms, but identifying therapeutic targets has remained challenging. Here, we integrate a two-cell type (2CT) whole-genome CRISPR-Cas9 screen with dynamic transcriptional profiling of the tumor/T cell interaction to comprehensively identify tumor genes that are induced to promote tumor survival. We assessed the therapeutic potential of pharmacological inhibition of these and other top CRISPR identified targets as combinatorial targets to improve the efficacy of tumor destruction by T cells.
Project description:<p>Due to the paucity of patient derived models in rare cancers, identification of therapeutic targets remains challenging. We developed a patient derived model, CLF-PED-015-T, from a patient with an undifferentiated sarcoma. From this model, we performed pooled RNAi and CRISPR-Cas9 negative selection screens and integrated that with a small molecule screen. Integration of these data identified CDK4 and XPO1 as potential therapeutic targets.</p>
