Programmable RNA-Guided Enzymes and Novel Orthogonal Substrates for Crispr Diagnostics

Reference Number TO 02-00434

Keywords

CRISPR-Cas, multiplex diagnosis, CRISPR substrates, molecular diagnostics

Invention Novelty

Programmable Cas12a2- and Cas12a3-based CRISPR diagnostics with truly orthogonal readouts for multiplex detection of ssDNA, dsDNA, or RNA.

Value Proposition

The new Cas12a2 and Cas12a3, and their specific orthogonal substrates, improve the speed and diagnostic specificity of current molecular diagnostics, and multiplex to at least 4 different nucleic acid sequences

Broad substrate versatility: Cas12a2: programmable target recognition across RNA, ssDNA, and dsDNA. Cas13a2: collateral activity that selectively cleaves tRNA (tRNA tail substrates), enabling an orthogonal reporter channel distinct from Cas12a2
Multiplexing: By using synthetic orthogonal substrates based on tRNA sequences, Cas12a3 achieves more than 4 different biomarkers per test, which can be expanded by using other Cas enzymes
Upstream or device need: Direct RNA detection without reverse transcription, enabling faster, simpler workflows

These features highlight the potential of synthetic orthogonal substrates for Cas12a2, and Cas12a3 to improve molecular diagnostic tests that can be used as point-of-care testing with low-complexity set-ups

Technology Description

Molecular Dx: Detection of RNA biomarkers in samples, particularly viral biomarkers (RSV, Influenza A and B)
Substrate: Cas12a2 preferentially targets ssDNA with additional functionality on dsDNA and RNA. Cas12a3 has a strong collateral activity toward tRNA, allowing clean and multiplex readouts without complexity
Cell arrest capabilities: tRNA tail cleavage induces transcription-dependent cell arrest without chromosomal DNA damage, allowing targeted therapeutics

No interference by human nucleic acid: Presence of human nucleic acid material enhances the reaction

Commercial Opportunity

The technology is available for co-development and in-licensing partnerships.

Development Status

Proof of concept shown.

Patent Situation

National phases with priority from 2021 have started in the US, Korea, Japan, Israel, Canada, China, Australia, and Europe for Cas12a2 (WO2022253903). A provisional priority application for the orthogonal substrates in the US was filed in November 2024.