To develop the method, we first designed sgRNAs targeting known resistance hotspots in genes sensitive to three cancer drugs: KPT-185, a preclinical analog of the XPO1 inhibitor selinexor16–19, ispinesib, an antineoplastic kinesin-5 (KIF11) inhibitor13,20, and triptolide, an antiproliferative agent targeting ERCC314,21 (Fig. 1a, b). The gene discussed is KIF11; the disease is cancer.