Electroporation-Based CRISPR/Cas9 Gene Editing Using Cas9 Protein and Chemically Modified sgRNAs

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas9 technology offers powerful genome editing capabilities.
• Transfection of certain human cancer cell lines and primary cells remains a significant challenge for gene editing.
• Conventional chemical-based transfection methods often show limited efficiency in hard-to-transfect cells.

Purpose of the Study:

• To develop and detail an efficient protocol for CRISPR/Cas9 delivery in challenging cell types.
• To overcome limitations of standard transfection methods for genome editing applications.
• To provide a reliable method for Cas9 protein and sgRNA delivery.

Main Methods:

• Electroporation-based delivery system.
• Utilized Streptococcus pyogenes Cas9 protein.
• Employed chemically modified single-guide RNAs (sgRNAs).
• Tested on various hard-to-transfect human cancer cell lines and primary cells.

Main Results:

• The protocol demonstrated high efficiency in delivering Cas9 protein and sgRNAs.
• Successfully achieved gene editing in cell lines and primary cells that are resistant to chemical transfection.
• Established a robust method for CRISPR/Cas9 application in previously intractable cellular models.

Conclusions:

• Electroporation offers a superior alternative for CRISPR/Cas9 delivery in difficult-to-transfect cells.
• This protocol enhances the accessibility of genome editing for a broader range of cancer research models.
• The method provides a valuable tool for advancing cancer cell line and primary cell research through precise gene editing.