Protocol for HiBiT tagging endogenous proteins using CRISPR-Cas9 gene editing.

ABSTRACT

We present a method of in vitro/in vivo protein detection by pairing CRISPR-Cas9 genome editing with the NanoBiT system. We describe steps for cell culturing, in vitro CRISPR-Cas9 ribonucleoprotein delivery, cell monitoring, efficiency assessments, and edit analysis through HiBiT assays. We then detail procedures to determine edit specificity through genomic DNA analysis, small interfering RNA reverse transfection, and HiBiT blotting. This protocol is simple to execute and multifunctional, and it enables high-throughput screens on endogenous proteins to be conducted with ease.