Compact RNA-guided DNA targeting, cleavage, and transcriptional activation

This technology is programmable, compact guide-RNA-directed DNA recognition using TnpB/Cas12-family effectors deployed for DNA cleavage or transcriptional repression/activation via DNA binding.

Unmet Need: Programmable compact gene regulators with high specificity

Current gene regulators, such as dCas9-based CRISPR interference/activation and viral transcription factor delivery, can enable programmable repression or activation of genes. However, these systems are large, delivery-limited, and often require complex fusion architectures, restricting their use in compact vectors and multiplexed systems. These systems also suffer from off-target binding and limited portability across organisms, highlighting the need for smaller, simpler, and more modular regulators.

The Technology: Compact RNA-guided DNA targeting

The technology is RNA-guided DNA targeting using compact Cas12/TnpB-family proteins that recognize specific genomic sites via gRNA base pairing and a short target-adjacent motif. Depending on the protein and engineering context, DNA binding can result in precise cleavage or be repurposed for transcriptional repression or activation without a double-strand break. In the activation mode, DNA binding recruits RNA polymerase through an associated sigma factor, enabling programmable transcription initiation at defined distances. This technology has been experimentally validated in E. coli.

Applications:

Programmable gene regulation in bacterial systems
Engineering of synthetic gene circuits and regulatory networks
High-throughput functional genomics in microbes
Metabolic pathway optimization through targeted transcriptional control
Precision bacterial genome engineering and reprogramming

Advantages:

Compact size enabling efficient delivery and multiplexing
Programmable RNA-guided DNA targeting without protein redesign
Minimal genomic disruption through non-cutting regulatory mechanisms
Simplified system architecture with fewer required components
Broad portability across diverse bacterial species

Lead Inventor:

Samuel Sternberg, Ph.D.

Patent Information:

Patent Pending (US 20250243514)