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 .
Keyphrases
- crispr cas
- genome editing
- randomized controlled trial
- copy number
- genome wide
- single cell
- cell therapy
- genome wide identification
- stem cells
- induced apoptosis
- high throughput
- cell proliferation
- genome wide analysis
- wild type
- mass spectrometry
- mesenchymal stem cells
- endoplasmic reticulum stress
- simultaneous determination