A Helicase-Fused Cas9 improves Large-Size Fragment Knock-in

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Genomics

Background:

• CRISPR/Cas9 systems are powerful gene editing tools, but large-size fragment knock-in (ls-KI) via homologous recombination (HR) remains inefficient in mammalian cells.
• Enhancing DNA repair pathways is crucial for improving the efficiency and accuracy of gene knock-in.

Purpose of the Study:

• To investigate whether incorporating DNA helicase activity into CRISPR/Cas9 can enhance ls-KI efficiency.
• To develop and characterize a novel gene editing tool, MCCas, for improved ls-KI.

Main Methods:

• Fusion of MCM5, a DNA helicase subunit, to the N-terminus of spCas9 to create the MCCas protein.
• Testing MCCas efficiency for ls-KI across multiple loci in human cell lines and rabbit embryos.
• Assessing on-target and off-target insertion/deletion (indel) events and mechanistic validation of the HR pathway.

Main Results:

• MCCas significantly increased ls-KI rates compared to spCas9 in human cells and rabbit embryos.
• MCCas enabled efficient ls-KI with large donor templates up to 10 kb.
• MCCas-mediated ls-KI showed reduced frequencies of on-target and off-target indel events.

Conclusions:

• The fusion of DNA helicase activity to CRISPR/Cas9 (MCCas) is a viable strategy to enhance ls-KI efficiency.
• MCCas represents a promising gene editing tool with improved capacity for large fragment knock-ins and reduced error rates.
• This advancement has significant implications for gene therapy, genetic engineering, and the development of disease models.