Engineering tripartite gene editing machinery for highly efficient non-viral targeted genome integration

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Genomics

Background:

• Non-viral DNA donor templates facilitate targeted genomic integration through homologous recombination (HR).
• CRISPR/Cas9 technology enhances HR efficiency for gene editing.
• Circular single-stranded DNA (cssDNA) acts as a genome engineering catalyst (GATALYST) for efficient and safe gene knock-in.

Purpose of the Study:

• To introduce enGager, an enhanced GATALYST associated genome editor system.
• To improve transgene integration efficiency using tethered cssDNA donors and modified Cas9.
• To evaluate enGager's efficacy in various cell types and for therapeutic gene delivery.

Main Methods:

• Tethering cssDNA donors to nuclear-localized Cas9 fused with single-stranded DNA binding peptide motifs.
• Utilizing the enGager system for targeted integration of reporter genes and a chimeric antigen receptor (CAR) transgene.
• Assessing integration efficiency in multiple genomic loci across various cell types, including primary human T cells.

Main Results:

• enGager significantly increased transgene integration efficiency, showing up to 6-fold higher efficiency compared to unfused Cas9.
• The system demonstrated improved targeted integration and expression of reporter genes.
• Efficient integration of a CAR transgene was achieved in 33% of primary human T cells, enhancing anti-tumor functionality.
• The 'tripartite editor with ssDNA optimized genome engineering (TESOGENASE) approach proved effective, especially for large transgenes in primary cells.

Conclusions:

• enGager enhances genome engineering efficiency and safety for targeted gene integration.
• The system provides a potent non-viral alternative to viral vectors for therapeutic gene modification.
• enGager shows promise for advancing gene-based therapies, including CAR T-cell therapy.