Improved specificity of CRISPR-associated transposon (CAST) systems

This technology is a genome engineering platform that enhances the specificity of CRISPR-associated transposase (CAST)-mediated DNA integration by modulating transposon protein activity to minimize off-target insertions.

Unmet Need: High-fidelity large DNA insertion without double-strand breaks

Current genome editing approaches that enable targeted DNA insertion, such as CRISPR-Cas nuclease-based systems, rely on double-strand DNA breaks and cellular repair pathways, which can lead to unpredictable outcomes, low efficiency, and genomic instability. CRISPR-associated transposases (CASTs) offer an alternative by enabling RNA-guided insertion of large DNA cargos without double-strand breaks. However, existing CAST systems, particularly Type V-K CASTs, exhibit poor insertion site specificity due to frequent RNA-independent transposition events. This lack of precision limits their utility in therapeutic genome engineering, functional genomics, and synthetic biology applications, where accurate and predictable DNA integration is essential.

The Technology: Mechanism-based enhancement of CAST insertion specificity

This technology improves the targeting accuracy of CRISPR-associated transposon systems by modulating the activity, abundance, and DNA-binding behavior of the transposon protein TnsC, a key determinant of insertion site selection. The engineered CAST systems retain the favorable properties of Type V-K CASTs, including compact size and programmable RNA-guided targeting, while achieving substantially improved on-target integration. The platform is compatible with multiple CAST subtypes and can be implemented through engineered protein variants, expression-level control, or modified system architectures.

This technology has been validated using in vitro and cellular assays, including sequencing-based analyses of insertion events, and is supported by structural and mechanistic studies as well as NIH-funded research and a published preprint.

Applications:

  • Genome engineering research tools for precise, RNA-guided insertion of large DNA payloads
  • Gene therapy development, including targeted gene addition without double-strand DNA breaks
  • Generation of engineered cell lines and animal models, such as knock-in systems for functional genomics
  • Synthetic biology and biopharmaceutical production, including microbial strain engineering
  • Agricultural genome modification, where controlled DNA integration is required
  • Sequencing and genomic analysis workflows involving RNA-guided DNA integration or tagmentation

Advantages:

  • Enables improved insertion site specificity compared to existing CRISPR-associated transposon systems
  • Reduces off-target DNA integration while preserving efficient RNA-guided insertion
  • Supports large DNA cargo integration without reliance on double-strand DNA breaks
  • Compatible with existing CRISPR-associated transposon architectures and workflows
  • Applicable across multiple CAST subtypes and experimental contexts
  • Provides more predictable and controllable genome engineering outcomes

Lead Inventor:

Samuel H. Sternberg, Ph.D.

Patent Information:

Patent Pending

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