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Updated: Aug 16, 2025
Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
Published on: February 26, 2019
An efficient and cost-effective purification protocol for Staphylococcus aureus Cas9 nuclease.
Allen C T Teng1, Marjan Tavassoli1, Suja Shrestha2
1Translational 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.
We developed a rapid and cost-effective protocol to purify Staphylococcus aureus Cas9 (SaCas9) protein with over 90% purity in 24 hours. This method accelerates CRISPR research by enabling easier access to functional SaCas9.
Area of Science:
- Molecular Biology
- Biotechnology
- Gene Editing Technologies
Background:
- Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) is a key enzyme in gene editing.
- Efficient purification of active Cas9 protein is crucial for advancing CRISPR-based research and therapeutic applications.
- Staphylococcus aureus Cas9 (SaCas9) offers unique advantages for certain CRISPR applications.
Purpose of the Study:
- To establish a streamlined protocol for the purification of functional SaCas9 protein.
- To achieve high purity and yield of SaCas9 within a short timeframe.
- To facilitate broader accessibility of SaCas9 for research laboratories.
Main Methods:
- The protocol involves a three-step purification process starting with immobilized metal affinity chromatography (IMAC).
- Subsequent purification steps include cation exchange chromatography (CEX).
- Final concentration and buffer exchange are performed using centrifugal concentrators.
Main Results:
- The developed protocol successfully purifies functional SaCas9 protein from Staphylococcus aureus.
- Achieved over 90% purity of SaCas9 within 24 hours.
- Demonstrated the simplicity, cost-effectiveness, and reproducibility of the purification method.
Conclusions:
- This protocol provides a rapid, efficient, and accessible method for obtaining high-purity SaCas9 protein.
- The widespread adoption of this protocol can significantly accelerate CRISPR research by lowering the barrier to Cas9 protein availability.
- Enables general laboratories to produce substantial amounts of SaCas9, fostering innovation in gene editing.
