Engineering Tripartite Gene Editing Machinery for Highly Efficient Non-Viral Targeted Genome Integration

Area of Science:

• Molecular Biology
• Gene Editing
• Biotechnology

Background:

• Non-viral DNA donor templates are crucial for targeted genomic integration via homologous recombination (HR).
• CRISPR/Cas9 systems have increased the efficiency of HR-mediated gene editing.
• Circular single-stranded DNA (cssDNA) has shown promise as a genome engineering catalyst (GATALYST) for safe and efficient gene knock-in.

Purpose of the Study:

• To develop an enhanced genome editor, enGager, that increases the integration efficiency of cssDNA donors.
• To evaluate the performance of enGager in site-directed genomic integration and transgene expression across various cell lines and genomic loci.
• To assess the potential of enGager for therapeutic gene modification, specifically for CAR-T cell engineering.

Main Methods:

• Development of enGager, a system tethering cssDNA donors to a nuclear-localized Cas9 fused with ssDNA binding peptides.
• Testing enGager's integration efficiency and expression of knocked-in genes (e.g., GFP) in multiple cell lines and genomic locations.
• Application of enGager for targeted integration of chimeric antigen receptor (CAR) transgene in primary human T cells.

Main Results:

• enGager significantly enhanced site-directed genomic integration efficiency, ranging from 1.5- to over 6-fold compared to unfused Cas9 editors.
• The enhancement was most pronounced for larger transgenes (> 4Kb) in primary cells, with a preference for non-concatemerized ssDNA donors.
• Efficient targeted integration of CAR transgene was achieved in 33% of primary human T cells, demonstrating functional anti-tumor activity.

Conclusions:

• The enGager system substantially improves the efficiency and safety of targeted gene integration using cssDNA donors.
• enGager overcomes limitations of viral vectors in terms of payload size and safety for therapeutic gene editing applications.
• The 'tripartite editors with ssDNA optimized genome engineering' (TESOGENASE™) system holds significant potential for advancing therapeutic gene modification.