A detailed protocol for expression, purification, and activity determination of recombinant SaCas9

Franziska Flottmann; Greta Marie Pohl; Jan Gummert; Hendrik Milting; Andreas Brodehl

doi:10.1016/j.xpro.2022.101276

A detailed protocol for expression, purification, and activity determination of recombinant SaCas9

STAR Protoc. 2022 Apr 5;3(2):101276. doi: 10.1016/j.xpro.2022.101276. eCollection 2022 Jun 17.

Authors

Franziska Flottmann¹, Greta Marie Pohl¹, Jan Gummert^{1

2}, Hendrik Milting¹, Andreas Brodehl¹

Affiliations

¹ Erich and Hanna Klessmann Institute, Heart and Diabetes Center NRW, University Hospital of the Ruhr-University Bochum, Georgstrasse 11, 32545 Bad Oeynhausen, Germany.
² Department of Thoracic and Cardiovascular Surgery, Heart and Diabetes Center NRW, University Hospital Ruhr-University Bochum, Georgstrasse 11, 32545 Bad Oeynhausen, Germany.

Abstract

Recombinant SaCas9 is useful for a broad range of applications in the context of genome editing, especially when the specific protospacer adjacent motifs of other Cas9 derivatives are missing. Here, we describe a detailed protocol for the expression and purification of recombinant SaCas9. We detail the main steps for immobilized metal affinity chromatography and size exclusion chromatography. In addition, we present an assay for activity determination of SaCas9. Active SaCas9 can be purified in a week by using this protocol.

Keywords: CRISPR; Protein Biochemistry; Protein expression and purification.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bacterial Proteins* / genetics
Gene Editing / methods
Staphylococcus aureus* / chemistry

Substances

Bacterial Proteins