An efficient and cost-effective purification protocol for Staphylococcus aureus Cas9 nuclease

Allen C T Teng; Marjan Tavassoli; Suja Shrestha; Ryan M Marks; Meghan J McFadden; Sonia L Evagelou; Kyle Lindsay; Ava Vandenbelt; Wenping Li; Evgueni Ivakine; Ronald Cohn; J Paul Santerre; Anthony O Gramolini

doi:10.1016/j.xpro.2022.101933

An efficient and cost-effective purification protocol for Staphylococcus aureus Cas9 nuclease

STAR Protoc. 2023 Mar 17;4(1):101933. doi: 10.1016/j.xpro.2022.101933. Epub 2022 Dec 26.

Authors

Affiliations

¹ Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research, University of Toronto, 661 University Avenue, 14th Floor, Toronto, ON M5G 1M1, Canada; Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address: allen.teng@utoronto.ca.
² Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research, University of Toronto, 661 University Avenue, 14th Floor, Toronto, ON M5G 1M1, Canada; Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada.
³ Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research, University of Toronto, 661 University Avenue, 14th Floor, Toronto, ON M5G 1M1, Canada; Faculty of Dentistry, University of Toronto, Toronto, ON M5G 1X3, Canada.
⁴ Department of Molecular Genetics, Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada; Program in Genetics and Genome Biology, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada.
⁵ Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research, University of Toronto, 661 University Avenue, 14th Floor, Toronto, ON M5G 1M1, Canada; Institute of Biomedical Engineering, University of Toronto, Toronto, ON M5S 3G9, Canada.
⁶ Program in Genetics and Genome Biology, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada.
⁷ Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research, University of Toronto, 661 University Avenue, 14th Floor, Toronto, ON M5G 1M1, Canada; Faculty of Kinesiology & Physical Education, University of Toronto, Toronto, ON M5S 2W6, Canada.
⁸ Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada; Program in Genetics and Genome Biology, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada.
⁹ Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research, University of Toronto, 661 University Avenue, 14th Floor, Toronto, ON M5G 1M1, Canada; Faculty of Dentistry, University of Toronto, Toronto, ON M5G 1X3, Canada; Institute of Biomedical Engineering, University of Toronto, Toronto, ON M5S 3G9, Canada.
¹⁰ Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research, University of Toronto, 661 University Avenue, 14th Floor, Toronto, ON M5G 1M1, Canada; Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address: anthony.gramolini@utoronto.ca.

Abstract

Here, we describe a protocol for purifying functional clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) from Staphylococcus aureus within 24 h and over 90% purity. SaCas9 purification begins with immobilized metal affinity chromatography, followed by cation exchange chromatography, and ended with centrifugal concentrators. The simplicity, cost-effectiveness, and reproducibility of such protocols will enable general labs to produce a sizable amount of Cas9 proteins, further accelerating CRISPR research.

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

Publication types

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

MeSH terms

CRISPR-Cas Systems* / genetics
Cost-Benefit Analysis
Gene Editing* / methods
Reproducibility of Results
Staphylococcus aureus / genetics