Protocol for establishing knockout cell clones by deletion of a large gene fragment using CRISPR-Cas9 with multiple guide RNAs

Genome editing is a powerful tool for establishing gene knockout or mutant cell lines. Here, we present a protocol for establishing knockout cell clones by deletion of large gene fragments using CRISPR-Cas9 with multiple guide RNAs. We describe steps for designing guide RNAs, cloning them into CRISPR-Cas9 vectors, cell seeding, transfection into cultured cells, clonal selection, and screening assays. This protocol can delete gene regions over 100 kbp, including GC-rich domains, and is applicable to various cell lines. For complete details on the use and execution of this protocol, please refer to Saito et al.,1 Saito and Endo et al.,2 and Higashi et al.3.