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

STAR Protocols (Mar 2023)

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

Affiliations

Allen C.T. Teng: 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; Corresponding author
Marjan Tavassoli: 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
Suja Shrestha: 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
Ryan M. Marks: 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
Meghan J. McFadden: 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
Sonia L. Evagelou: Program in Genetics and Genome Biology, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada
Kyle Lindsay: Program in Genetics and Genome Biology, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada
Ava Vandenbelt: 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
Wenping Li: 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
Evgueni Ivakine: 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
Ronald Cohn: 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
J. Paul Santerre: 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
Anthony O. Gramolini: 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; Corresponding author

Journal volume & issue: Vol. 4, no. 1
p. 101933

Abstract

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Summary: 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. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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