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.
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.
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.
Patent Pending
IR CU24013
Licensing Contact: Cynthia Lang
