Controlled and specific RNA-guided DNA integration

This technology is a method that utilizes Tn7 transposons to facilitate site-specific genome integration, eliminating off-target effects in gene editing.

Unmet Need: Genome editing with reduced off-target effects

Current methods of CRISPR/Cas9-based gene editing rely on site-specific double-stranded breaks (DSBs) in the genome to target a gene of interest. This DSB is repaired by non-homologous end joining, or homologous recombination with a repair fragment. However, these processes are not without problems. Non-homologous end joining can limit the efficiency of DNA integration and lead to off-target effects; furthermore, not all cells possess the machinery necessary to facilitate homologous recombination. These off-target effects result in heterogeneity in gene-edited cells, which limits the future application of genome editing therapies.

The Technology: Transposase system for site-specific integration

This technology describes an RNA-guided transposase system that utilizes Tn7 transposons to integrate a DNA fragment with minimal off-target mechanisms. Specific site integration can be guided by altering the RNA spacer sequence. This technology significantly reduces the off-target effects of genome editing compared to integration techniques such as non-homologous end joining or homologous recombination.

Proof-of-concept experiments show efficient integration into the intergenic regions of three different genes in cells.

Applications:

  • Delivery of multi-gene circuits or synthetic genes into different genomes
  • Gene therapy for genetic diseases
  • Gene editing of CAR-T cells for cancer immunotherapy
  • Research tool for genome-wide screening using guide RNA libraries
  • Research tool for gene essentiality studies

Advantages:

  • Increases safety by decreasing off-target effects
  • Increases insertion efficiency
  • Enables controlled and site-specific genome integration
  • Ease-of-use with simple RNA engineering and editing
  • Eliminates risks associated with double-stranded breaks

Lead Inventor:

Sam Sternberg, Ph.D.

Patent Information:

Patent Issued (US 12,331,292)

Related Publications:

Tech Ventures Reference: