ArticleMethods in molecular biology (Clifton, N.J.)2027
Production and Purification of Cas9 Protein for Ribonucleoprotein Complex Formation.
Article in Methods in molecular biology (Clifton, N.J.), 2027. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
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.
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