CRISPR/Cas9 Ribonucleoprotein Delivery Enhanced by Lipo-Xenopeptide Carriers and Homology-Directed Repair Modulators:...

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• CRISPR-Cas9 is a powerful tool for disease research and treatment.
• Homology-directed repair (HDR) is crucial for gene correction but faces efficiency challenges.
• Developing effective delivery systems and enhancers is vital for therapeutic gene editing.

Purpose of the Study:

• To investigate strategies for enhancing gene correction efficiency using CRISPR-Cas9.
• To evaluate the efficacy of a T-shaped lipo-xenopeptide (XP)-based delivery system combined with HDR enhancers.
• To identify novel enhancers for CRISPR-Cas9 mediated gene editing.

Main Methods:

• Utilized a Cas9 RNP/ssDNA delivery system with various HDR enhancers.
• Tested Nu7441, a DNA-PKcs inhibitor, for its effect on HDR efficiency in HeLa-eGFPd2 cells.
• Assessed gene editing efficiency, including NHEJ, in reporter cell models and various cell lines.

Main Results:

• Nu7441 demonstrated a significant enhancement of HDR-mediated gene correction, achieving over 10-fold increase and up to 61% efficiency.
• Nu7441 also boosted NHEJ-mediated exon skipping by 30-fold in a DMD reporter model.
• The enhancer was effective across multiple cell lines and delivery systems, including Cas9 mRNA polyplexes.

Conclusions:

• Nu7441 acts as a potent enhancer for both HDR and total gene editing efficiency with CRISPR-Cas9.
• The study presents a novel strategy to improve Cas9 RNP-based gene therapy outcomes.
• Nu7441's mechanism involves cell cycle modulation without compromising delivery efficiency.