Versatile Cell Penetrating Peptide for Multimodal CRISPR Gene Editing in Primary Stem Cells

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Cellular Delivery Systems

Background:

• CRISPR gene editing requires efficient and safe delivery of its components into target cells.
• Current viral and non-viral delivery methods face challenges including cytotoxicity, immunogenicity, and limited cargo versatility.
• The arginine-alanine-leucine-alanine (RALA) peptide is a cell-penetrating peptide known for low cytotoxicity and effective delivery of various molecules.

Purpose of the Study:

• To establish the RALA peptide as a novel, safe, and versatile delivery system for CRISPR gene editing components.
• To evaluate RALA's efficacy in delivering CRISPR in DNA, RNA, and ribonucleoprotein (RNP) formats to primary mesenchymal stem cells (MSCs).
• To compare RALA's performance against existing commercial reagents regarding cell viability, transfection rates, and cell function maintenance.

Main Methods:

• Utilized the RALA peptide to encapsulate and deliver CRISPR-Cas9 components in DNA, RNA, and RNP formats.
• Transfected primary human mesenchymal stem cells (MSCs) with RALA-delivered CRISPR systems.
• Assessed cell viability, transfection efficiency, and cell proliferative capacity post-delivery.
• Performed gene knock-in, knock-out, and transcriptional activation using RALA-mediated CRISPR delivery.

Main Results:

• RALA effectively encapsulated and delivered CRISPR components in all tested formats (DNA, RNA, RNP) to MSCs.
• RALA demonstrated superior cell viability and higher transfection rates compared to commercially available reagents.
• RALA delivery maintained the proliferative capacity of MSCs.
• Successful gene editing outcomes including reporter gene knock-in/knock-out and transcriptional activation were achieved.

Conclusions:

• RALA serves as a powerful and safe non-viral tool for delivering CRISPR gene editing machinery.
• The RALA peptide system offers versatility across multiple CRISPR cargo formats and editing strategies.
• RALA enhances gene editing efficiency and cell viability, presenting a promising alternative for therapeutic applications.