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

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.