Target identification with systematic CRISPR-based chemical-genetic profiling in human HAP1 cells

我们建议在人类HAP1细胞系（源自白血病细胞系的近单倍体细胞系）中建立并应用全基因组化学基因组相互作用筛选平台，以功能表征化合物并阐明其在人类细胞中的靶点。我们独特和创新的方法将导致建立一个参考人类化学-遗传相互作用数据库，用于已知作用模式的化合物，以及RIKEN天然产物库（NPDepo）或东京大学药物发现倡议（DDI）核心库中先前未表征的化合物的功能注释。我们将生化验证一系列有前途的工具化合物的作用，这些化合物可用于探索细胞生物学的各个方面，并被开发为药物开发的潜在线索。在FY2022，我们使用并建立了一个独特的CRISPR-Cas9 gRNA文库，即Toronto KnockOut文库v3，涵盖了约18,000个人类基因，从而实现了哺乳动物细胞全基因组规模的功能缺失筛选。为了选择在人类化学基因组学管道中进行检测的生物活性化合物，我们筛选了RIKEN NPDepo和东京大学DDI核心文库中基于我们集成的酵母化学遗传平台具有强大且高度保守的目标预测的化合物。

We proposed to build and apply a genome-wide chemical genomic interaction screening platform in a human HAP1 cell line (a near-haploid cell line derived from a leukemic cell line), to functionally characterize compounds and elucidate their targets in human cells. Our unique and innovative approach will lead to the establishment of a reference human chemical-genetic interaction database for compounds with known modes of action, and the functional annotation of previously uncharacterized compounds from the RIKEN Natural Product Depository (NPDepo) or the University of Tokyo Drug Discovery Initiative (DDI) Core Library. We will biochemically validate roles of a collection of promising tool compounds that can be used to explore diverse aspects of cell biology and exploited as potential leads for drug development. In FY2022, we employed and set up a unique CRISPR-Cas9 gRNA library, the Toronto KnockOut library v3, covering ~18,000 human genes to enable genome-wide scale loss of function screens in mammalian cells. To select bioactive compounds to be examined in the human chemical genomics pipeline, we screened the RIKEN NPDepo and the University of Tokyo DDI Core Library for the compounds that possess strong and highly conserved target predictions based on our integrated yeast chemical-genetic platform.