ptARgenOM-A Flexible Vector For CRISPR/CAS9 Nonviral Delivery

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Viral delivery of CRISPR-Cas9 is common for gene function analysis but has limitations.
• Intracellular protein studies and selection of knockout (KO) cells can be laborious.
• Viral vectors introduce unwanted genetic material, causing experimental bias.

Purpose of the Study:

• To present a non-viral, all-in-one CRISPR-Cas9 delivery system for efficient generation of KO polyclonal cells.
• To overcome the limitations of viral delivery methods in genome editing.
• To provide a faster and more cost-effective tool for creating isogenic KO cell lines.

Main Methods:

• Development of the ptARgenOM mammalian expression vector for CRISPR-Cas9 delivery.
• The vector encodes Cas9, gRNA, and selection markers (eGFP, puromycin N-acetyltransferase) linked by a ribosomal skipping sequence.
• Transient, expression-dependent selection and enrichment of isogenic KO cells.

Main Results:

• ptARgenOM demonstrated efficiency in generating KO cells across diverse targets and cell lines.
• The system significantly reduced the time to obtain polyclonal isogenic cell lines by 4-6 fold.
• Non-viral delivery avoided the integration of unwanted genetic material.

Conclusions:

• ptARgenOM offers a simple, fast, and cost-effective alternative for genome editing.
• This method facilitates efficient selection and enrichment of polyclonal KO cells.
• The tool streamlines the process of analyzing gene function through genome editing.