Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches

Area of Science:

• Biotechnology
• Genetics
• Molecular Biology

Background:

• Classical gene therapy (GT) uses lentivirus (LV) or adeno-associated virus (AAV) for transgene delivery, risking severe side effects due to random genome integration.
• CRISPR/Cas9 gene editing enables precise transgene insertion via homologous direct repair (HDR), a process often limited by cellular mechanisms favoring non-homologous end-joining (NHEJ).

Purpose of the Study:

• To review current strategies for enhancing HDR efficiency in CRISPR/Cas9-based gene therapy.
• To highlight the advantages of CRISPR/Cas9 genome editing over classical GT approaches.

Main Methods:

• Comprehensive literature review of existing methods to improve HDR efficiency.
• Analysis of CRISPR/Cas9 system mechanisms and cellular repair pathways (HDR vs. NHEJ).

Main Results:

• Classical GT methods (LV, AAV) have demonstrated severe adverse events in clinical trials.
• CRISPR/Cas9 requires efficient HDR for precise gene insertion, but NHEJ often predominates.
• Various approaches are being explored to boost HDR efficiency for safer and more effective gene therapy.

Conclusions:

• Improving HDR efficiency is critical for advancing CRISPR/Cas9-based gene therapy.
• CRISPR/Cas9 holds significant promise for precise gene therapy, provided HDR can be effectively harnessed.
• Further research into HDR enhancement is essential for overcoming current gene therapy challenges.