Self-amplifying, in-field RNA detection platform using type III-D CRISPR technology

Overview

This solution offers a novel RNA detection platform built around a Type III-D CRISPR system, enabling highly sensitive and specific in-field detection of RNA transcripts without requiring complex pre-amplification steps. The platform is particularly suited for agricultural applications, such as verifying gene edits in plant genomes. By exploiting the innate signal amplification properties unique to Type III CRISPR systems, the assay achieves femtomolar-level sensitivity while keeping costs low and simplifying workflow. The platform supports multiple readout methods—including fluorescent, chemical, and lateral flow—making it adaptable to diverse field and laboratory environments. Its potential tolerance of lyophilisation may further enable shelf-stable, ambient-temperature storage.

Technical specifications

• Innate signal amplification: Type III CRISPR systems produce cyclic oligoadenylates (cOAs) upon target RNA recognition, which activate accessory nucleases that cleave reporter molecules—creating two built-in isothermal amplification steps.
• No pre-amplification required: Achieves femtomolar sensitivity without LAMP, RPA, or other pre-amplification methods, simplifying the workflow and reducing equipment needs.
• Type III-D advantage: Fewer subunits than other Type III systems, which may preserve enzymatic function after lyophilisation and enable ambient storage.
• Flexible readout: Compatible with fluorescent, chemical, or lateral flow detection methods.
• Simple re-targeting: New RNA targets can be detected by simply changing the guide RNA.
• Crude sample compatibility: Designed to function with rapid, simple sample preparation suitable for in-field testing.
• Low cost of goods: Components are expressed in bacterial systems and required in low amounts, targeting a per-test cost below $10.
• Rapid detection: Fluorescence signal observable within 30 minutes at 30°C.

Technology readiness level

The Type III-Dv enzyme has been successfully expressed, purified, and biochemically characterised. Proof-of-concept validation has confirmed specific detection of target RNA via fluorescent reporter cleavage, with signal observable within 30 minutes at 30°C. Next steps include assay optimisation with mutant enzyme variants, sensitivity testing with synthetic and plant-extracted RNA, evaluation of rapid extraction/lysis methods, lyophilisation stability testing, and development of visual readout formats such as lateral flow. The platform is at an early-to-mid stage of development, with demonstrated biochemical feasibility progressing toward field-ready assay validation.