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A One-Step Method for Efficient Purification of Functional Cas9 Protein.

Xinzhi Duan1,2, Zhengrong Zhou2, Aihua Mao1,2

  • 1Biology Department, College of Sciences, Shantou University, Shantou, China.

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|February 12, 2026
PubMed
Summary

We developed a simplified, one-step CRISPR/Cas9 purification method using a ubiquitin fusion system in E. coli. This approach yields high-purity, functional Cas9 protein for genome editing applications.

Keywords:
Affinity chromatographyGene editingProtein purificationSpCas9Ubiquitin-fusion

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetics

Background:

  • CRISPR/Cas9 is vital for genome editing.
  • Cas9 ribonucleoprotein (RNP) complexes offer advantages like reduced off-target effects.
  • Current Cas9 purification methods are complex, time-consuming, and reduce yield.

Purpose of the Study:

  • To develop a simplified, high-yield purification strategy for functional Streptococcus pyogenes Cas9 (SpCas9).
  • To utilize a ubiquitin (Ub) fusion system in Escherichia coli for efficient SpCas9 production.
  • To enable direct purification of active Cas9 for genome editing applications.

Main Methods:

  • Employed an N-terminal ubiquitin fusion strategy for His-Ub-Cas9 in E. coli.
  • Utilized nickel-affinity chromatography for single-step purification, eliminating protease cleavage.
  • Validated the activity of purified His-Ub-Cas9 in zebrafish embryos.

Main Results:

  • Achieved high-yield production (>8 mg/L) of >95% pure His-Ub-Cas9 fusion protein.
  • Demonstrated that the Ub tag does not require proteolytic removal for purification.
  • Confirmed robust DNA cleavage activity and genome editing efficiency of the purified protein in vivo.

Conclusions:

  • The ubiquitin fusion system provides a simplified, one-step method for producing functional SpCas9.
  • This streamlined protocol facilitates the broad application of Cas9 protein in genome editing.
  • The method enhances protein solubility and yield, making Cas9 readily available for RNP complex formation.