Long-offset paired nicking-based efficient and precise strategy for in vivo targeted insertion

Area of Science:

• Gene editing and therapy
• Molecular biology
• Genomics

Background:

• CRISPR-Cas9 technology offers potential for gene therapy through targeted DNA insertion.
• Efficient and precise integration of large DNA fragments in somatic cells remains a significant challenge.
• Current methods face difficulties in avoiding unintended genetic modifications.

Purpose of the Study:

• To develop a novel strategy for enhanced targeted gene integration in somatic cells.
• To improve the efficiency and precision of knock-in (KI) compared to existing nickase-based methods.
• To assess the efficacy of the new strategy in a relevant disease model.

Main Methods:

• Devised a Long-Offset Paired Nicking Target Integration (LOTI) strategy utilizing Cas9 nickase (Cas9n).
• Investigated the involvement of DNA repair proteins Rad51, Rad52, and Ligase I/III in the repair mechanism.
• Tested LOTI for integrating large gene fragments (≥1.5 kb) in hepatocytes and in a hemophilia B mouse model.

Main Results:

• LOTI demonstrated higher knock-in efficiency in somatic cells than classical nickase approaches.
• Efficient integration of at least 1.5-kb gene fragments was achieved.
• Restored 55% of Factor IX (FIX) activity in a hemophilia B mouse model with a single plasmid DNA dose.
• LOTI significantly reduced off-target mutations and unwanted insertions/deletions (indels) at the target site.

Conclusions:

• The LOTI strategy provides a precise and efficient method for targeted gene integration in somatic cells.
• This approach enhances the utility of CRISPR-based gene editing for therapeutic applications.
• LOTI offers a promising tool for in vivo gene therapy, minimizing unintended genetic alterations.