Production and Purification of Cas9 Protein for Ribonucleoprotein Complex Formation.

Cas9-guide RNA ribonucleoprotein (RNP) complexes are widely used for genome editing because they provide rapid, transient nuclease activity without introducing exogenous DNA into target cells. Efficient RNP delivery requires a highly purified, correctly folded Cas9 protein that is free of contaminating nucleases and small-molecule impurities. This chapter describes the expression of N-terminally His-tagged Cas9 from a pET28b vector in Escherichia coli Rosetta strains, followed by purification using Ni-NTA immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) on a Superdex 200 Increase column. The protocol covers bacterial fermentation, cell lysis, IMAC with high-salt and graded-imidazole washes, SEC polishing, dialysis into a storage buffer, and concentration to RNP-ready stocks, with quality control by SDS-PAGE and optional western blotting and BCA assay. Finally, a short procedure for assembling Cas9 RNPs with in vitro-transcribed guide RNAs is provided, making the protocol directly applicable to CRISPR RNP delivery in mammalian cells.