DsDNA-templated Substrates as Fluorescent Probes for CRISPR-Cas12a-based Diagnostics

Background:

Scientists use the naturally occurring CRISPR-Cas12a gene editing system as a valuable tool for disease diagnostics. When Cas12a finds its target -- a sequence of double-stranded DNA -- it cuts it. In the process, it also cuts other nearby DNAs. Scientists program Cas12a to find a particular disease-causing genetic sequence, then pair it with single-stranded DNA that's been genetically modified to fluoresce when cut. When Cas12a finds and cuts its target, it also cuts the genetically modified DNA. The fluorescent "probe" thus signals that Cas12a has found its target. This disclosure reports that Cas12a cleavage can be extended to double-stranded DNA, expanding biochemical options for DNA-based fluorescent probes. The CRISPR gene editing system enables scientists to locate disease-causing genetic sequences. A new CRISPR-based technology developed by researchers at the University at Albany offers improved signaling probes for disease diagnostics in cancer testing, agricultural screening and food safety.

Technology Overview:

Cas12a cleavage can be extended to double-stranded DNA to create probes with low noise, lower cost, higher yield and fast fluorescence recovery rate. Fluorescence depends on the distance between the molecules that control it (F and Q). With double-stranded DNA, the length of the probe can be changed indefinitely without altering the distance between F and Q. Single-stranded DNA probes have limited options, because as the probe length increases, the spacing between F and Q (hence noise) increases. Using double-stranded DNA to create fluorescent probes opens a broader biochemical space for designing improved DNA-based fluorescent probes for cas12a-based diagnostics.

Advantages:

•    Low noise
•    Lower cost
•    Higher yield
•    Fast fluorescence recovery rate

Applications:

•    Cancer testing
•    Agricultural screening
•    Food safety

Intellectual Property Summary:

US Patent Pending, Application No: 17/710,673 (filed 3/31/2022)

Licensing Status:

This technology is available for licensing.