Functional DNA Regulated CRISPR-Cas12a Sensors for Point-of-Care Diagnostics of Non-Nucleic-Acid Targets

Beyond its extraordinary genome editing ability, the CRISPR-Cas systems have opened a new era of biosensing applications due to its high base resolution and isothermal signal amplification. However, the reported CRISPR-Cas sensors are largely only used for the detection of nucleic acids with limited application for non-nucleic-acid targets. To realize the full potential of the CRISPR-Cas sensors and broaden their applications for detection and quantitation of non-nucleic-acid targets, we herein report CRISPR-Cas12a sensors that are regulated by functional DNA (fDNA) molecules such as aptamers and DNAzymes that are selective for small organic molecule and metal ion detection. The sensors are based on the Cas12a-dependent reporter system consisting of Cas12a, CRISPR RNA (crRNA), and its single-stranded DNA substrate labeled with a fluorophore and quencher at each end (ssDNA-FQ), and fDNA molecules that can lock a DNA activator for Cas12a-crRNA, preventing the ssDNA cleavage function of Cas12a in the absence of the fDNA targets. The presence of fDNA targets can trigger the unlocking of the DNA activator, which can then activate the cleavage of ssDNA-FQ by Cas12a, resulting in an increase of the fluorescent signal detectable by commercially available portable fluorimeters. Using this method, ATP and Na⁺ have been detected quantitatively under ambient temperature (25 °C) using a simple and fast detection workflow (two steps and <15 min), making the fDNA-regulated CRISPR system suitable for field tests or point-of-care diagnostics. Since fDNAs can be obtained to recognize a wide range of targets, the methods demonstrated here can expand this powerful CRISPR-Cas sensor system significantly to many other targets and thus provide a new toolbox to significantly expand the CRISPR-Cas system into many areas of bioanalytical and biomedical applications.