Abstract
Cas9 is a site-specific RNA-guided endonuclease (RGEN) that can be used for precise genome editing in various cell types from multiple species. Ribonucleoprotein (RNP) complexes, which contains the Cas9 protein in complex with a guide RNA, are sufficient for the precise editing of genomes in various cells. This DNA-free method is more specific in editing the target sites and there is no integration of foreign DNA into the genome. Also, there are ongoing studies into the interactions of Cas9 protein with modified guide RNAs, as well as structure-activity studies of Cas9 protein and its variants. All these investigations require highly pure Cas9 protein. A single-step metal affinity enrichment yielding impure Cas9 is the most common method of purification described. This is sufficient for many gene editing applications of this protein. However, to obtain Cas9 of higher purity, which might be essential for biophysical characterization, chemical modifications, and structural investigations, laborious multi-step protocols are employed. Here, we describe a two-step Cas9 purification protocol that uses metal affinity enrichment followed by cation exchange chromatography. This simple method can yield a milligram of highly pure Cas9 protein per liter of culture in a single day.
Highlights
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is found in bacteria and archaea and act as their adaptive immunity against invading foreign nucleic acids [1]
For higher purity of Cas9 additional purification, steps like cation exchange chromatography and size exclusion chromatography have been added to the initial affinity enrichment [1,6]
We have developed a simple two-step Cas9 purification protocol that yielded milligrams of highly pure protein
Summary
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is found in bacteria and archaea and act as their adaptive immunity against invading foreign nucleic acids [1]. A simple and rapid protocol for obtaining pure nuclease protein is essential for these biophysical, chemical modification, and structural biology efforts. For genome editing techniques and in vitro nuclease assays, Cas is purified by a single-step metal affinity enrichment [3,4,5]. We have developed a simple two-step Cas purification protocol that yielded milligrams of highly pure protein. We achieved this by adding a high-resolution cation exchange chromatography step right after the affinity enrichment of Cas. We achieved this by adding a high-resolution cation exchange chromatography step right after the affinity enrichment of Cas9 Using this protocol, we were able to purify Cas to near homogeneity within a few hours
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