Chimeric CRISPR-associated transposon system for RNA-mediated DNA integration

This technology is a chimeric CRISPR-associated transposon (CAST) system that allows for more efficient and specific RNA-guided DNA integration.

Unmet Need: Efficient RNA-guided DNA integration system without DNA double-strand breaks

Current methods for genome editing rely heavily on DNA double-stranded breaks and homologous recombination to facilitate DNA insertions, which can reduce insertion efficiency, promote toxicity, and lead to off-target effects. Additional approaches for DNA integration are being developed using CRISPR-associated transposase (CAST) systems, which use guide RNAs to direct a transposase enzyme to a specific site for catalysis of the DNA insertion without a double-strand break. While these CAST systems are capable of inserting larger DNA cargo, they are limited by a lack of efficiency and the inability to combine components of different CAST systems into one due to the co-evolution of the protein complexes within each system. Therefore, there is a need for RNA-mediated DNA integration with improved efficiency and specificity without the use of DNA double-strand breaks.

The Technology: Mixed-origin CAST system for efficient large DNA cargo integration

This technology utilizes engineered components from previously orthogonal CAST systems to create a chimeric CAST system that optimizes each step of the RNA-guided DNA integration process, even in the absence of DNA double-strand breaks. By modifying the structure of different CAST components, this technology has improved DNA recognition capabilities, stabilized protein-RNA and protein-protein interactions, and introduced flexible protospacer adjacent motif (PAM) requirements. This technology demonstrates that additional combinations of CAST components can be used to further enhance this method of human genome editing. This technology can be used to generate knock-in cell lines and animal models, as well as human therapies and engineered crops.

This technology has been validated in human cell lines.

Applications:

  • Generation of knock-in cell lines and animal models
  • Generation of engineered crops
  • Research tools for studying DNA integration
  • Human gene therapeutics

Advantages:

  • High efficiency and specificity of DNA insertions
  • DNA insertions without double-strand breaks
  • Large cargo insertion
  • No dependence on homologous recombination machinery
  • Ability to use components from multiple CAST systems

Lead Inventor:

Samuel Sternberg, Ph.D.

Patent Information:

Patent Pending

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