Compact bacterial recombination complexes drive efficient kilobase-scale knock-in in mammalian cells

Area of Science:

• Molecular Biology
• Genetics
• Gene Editing Technologies

Background:

• Efficient homology-directed repair (HDR) is crucial for precise genome editing, especially for large DNA insertions in mammalian cells.
• The utility of bacterial recombineering proteins, like RecE and RecT, in eukaryotic systems for enhancing HDR remains largely unexplored.

Purpose of the Study:

• To investigate the potential of bacterial recombineering proteins to enhance HDR efficiency in mammalian cells.
• To develop novel tools for improving kilobase-scale DNA insertions via HDR for precise genome engineering.

Main Methods:

• Identification and characterization of Escherichia coli RecE (EcRecE) for its role in enhancing HDR in mammalian cells.
• Targeted recruitment of EcRecE using CRISPR/Cas9 systems to facilitate HDR at various genomic loci.
• Development of a dCas9-miniRecTE editor for large-fragment integration without inducing double-strand breaks.

Main Results:

• EcRecE significantly enhanced HDR efficiency, achieving a 3-6 fold increase in kilobase-scale sequence integration in human cells.
• The dCas9-miniRecTE editor demonstrated approximately 20% efficiency for kilobase-scale knock-in in human cells and primary neurons.
• Demonstrated successful application across different cell types, including human embryonic stem cells.

Conclusions:

• Escherichia coli RecE (EcRecE) is identified as a potent enhancer of homology-directed repair in mammalian cells.
• The developed dCas9-miniRecTE system offers a novel, double-strand break-free method for large-fragment genome editing.
• These findings provide versatile tools for advancing precision genome engineering and hold promise for therapeutic gene editing applications.