ARTICLE | Distillery Techniques

Assays and screens

March 20, 2018 11:14 PM UTC

CRISPR-based screening in human cells could help identify the therapeutic targets of small molecules. The method involved three steps: generating in-frame mutations in human cell lines by engineering them to express CRISPR-associated protein 9 (Cas9) or CRISPR from Prevotella and Francisella 1 (Cpf1), plus a single-guide RNA (sgRNA) library targeting the corresponding Cas9 or Cpf1 protospacer adjacent motif (PAM) sites within genomic protein-coding regions; treating the cells with a small molecule agent; and sequencing sgRNAs and their corresponding targets in surviving cells to identify genes with resistance-causing mutations, whose corresponding wild-type could be the small molecule’s target. In a human chronic myelogenous leukemia (CML) cell line expressing Cpf1 and a sgRNA library targeting all Cpf1 PAM sites in 10 cancer-related genes, the method correctly identified exportin 1 (XPO1; CRM1) as the target of the XPO1 inhibitor selinexor. In two human CML cell lines expressing Cas9 and a sgRNA library targeting all Cas9 PAM sites in 75 cancer-related genes, the method identified nicotinamide phosphoribosyltransferase (NamPRT; NAMPT) as an additional target of the p21 protein-activated kinase 4 (PAK4) inhibitor KPT-9274. Next steps include developing larger sgRNA libraries for the method.

Karyopharm Therapeutics Inc. and Ono Pharmaceutical Co. Ltd. have selinexor (KPT-330) in Phase I through Phase III testing to treat various cancers...