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:This is data for the evaluation of a new way of counting sgRNAs in CRISPR screens using padlock probes and UMIs. It is compared to the typical PCR-based approach. In particular, a dropout screen was performed in MiaPaCa-2 cells using the Human Kinome CRISPR pooled library (Addgene #75314)
Project description:The goal of the project is to identify deubiquitinases (DUBs) networks that could be targetable in cancer. To identify functionally interacting DUBs, we performed a pooled combinatorial knockout screen using a CRISPR/Cas12a system. Here we transduced a custom guide library targeting any 2-gene combination derived from 160 genes (including ~90 DUBs and other genes of interest) into Cas12a-expressing HCT116 cells. While the cells are subjected to puromycin selection, a reference sample (Day 4 post-transduction) was taken and sequenced. Two replicates were derived from this pool that were expanded and sequenced at Day 11 and Day 18 post-transduction.
Project description:Current protein engineering methods are inadequate to explore the combinatorial potential offered by nature’s vast repertoire of protein domains – limiting our ability to create optimal synthetic tools. To overcome this barrier, we develop an approach to create and test thousands of chimeric proteins and employ it to probe an expansive combinatorial landscape of over 15,000 multi-domain CRISPR activators. Our findings indicate that many activators produce substantial cellular toxicity, often unrelated to their capacity to regulate gene expression. We also explore the biochemical features of activation domains and determine how their combinatorial interactions shape activator behavior. Finally, we identify two potent CRISPR activators, MHV and MMH, and demonstrate their enhanced activity across diverse targets and cell types compared to the gold-standard MCP activator, synergistic activation mediator (SAM)
