Advancement in Gene Editing: Efficient Integration of Large Genes in Mammalian Cells from Broad Institute

A recent study published in Nature Biomedical Engineering presents a new method for more efficient and precise integration of large genes into mammalian genomes. The research, led by Smriti Pandey, Xin D. Gao, and their team, introduces prime-editing-assisted site-specific integrase gene editing (PASSIGE). This technique aims to enhance gene therapy and genetic research.

Addressing Gene Integration Challenges
Current methods for targeted gene integration in mammalian cells often struggle with issues such as low efficiency and specificity. Techniques like CRISPR-associated transposases and programmable nucleases can cause unintended effects, including double-strand breaks and uncontrolled insertions.

The PASSIGE Approach
PASSIGE combines prime editing and site-specific recombinases to integrate large DNA sequences into specific genomic sites efficiently. The researchers enhanced this process using phage-assisted continuous evolution (PACE) to optimize recombinase activity.

Improved Recombinases
The team focused on evolving the Bxb1 recombinase, creating variants named evoBxb1 and eeBxb1. These variants showed significant improvements in gene integration efficiency:

• EvoBxb1: Achieved a 2.7-fold increase in efficiency.
• EeBxb1: Showed a 4.2-fold improvement.

Enhanced Efficiency
In human cell lines with pre-installed recombinase landing sites, evoBxb1 and eeBxb1 achieved donor integration rates up to 60%, much higher than the wild-type Bxb1. This resulted in targeted gene integration efficiencies averaging 23%, and over 30% in primary human fibroblasts.

Comparison with Existing Methods
PASSIGE with evoBxb1 or eeBxb1 outperformed the PASTE method (programmable addition via site-specific targeting elements) by 9.1-fold and 16-fold, respectively, demonstrating its effectiveness for targeted gene integration in mammalian cells.

Future Potential
This method’s ability to precisely and efficiently integrate large genes into mammalian genomes could significantly impact treatments for genetic diseases. By preserving physiological gene expression, it avoids complications associated with viral vector-mediated gene therapy.

Conclusion
The development of PASSIGE with continuously evolved recombinases represents a notable advancement in genetic engineering. This technology has the potential to improve gene therapies, offering new possibilities for patients with genetic disorders.

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