A protospacer adjacent motif-free, multiplexed, and quantitative nucleic acid detection platform with barcode-based Cas12a activity

Clustered regularly interspaced short palindromic repeat (CRISPR)-based biosensors have been developed to facilitate the rapid and sensitive detection of nucleic acids. However, most approaches using CRISPR-based detection have disadvantages associated with the limitations of CRISPR RNA (crRNA), protospacer adjacent motif (PAM) or protospacer flanking sequence restriction, single channel detection, and difficulty in quantitative detection resulting in only some target sites being detected qualitatively. Here, we aimed to develop a barcode-based Cas12a-mediated DNA detection (BCDetection) strategy, which overcomes the aforementioned drawbacks and enables (1) detection with a universal PAM and crRNA without PAM or crRNA restriction, (2) simultaneous detection of multiple targets in a single reaction, and (3) quantitative detection, which can significantly distinguish copy number differences up to as low as a two-fold limit. We could efficiently and simultaneously detect three & beta;-thalassemia mutations in a single reaction using BCDetection. Notably, samples from normal individuals, spinal muscular atrophy (SMA) carriers, and SMA patients were significantly and accurately distinguished using the quantitative detection ability of BCDetection, indicating its potential application in & beta;-thalassemia and SMA carrier screening. Therefore, our findings demonstrate that BCDetection provides a new platform for accurate and efficient quantitative detection using CRISPR/Cas12a, highlighting its bioanalytical applications.

Automated summary

In this study, a CRISPR-based biosensor was developed for sensitive detection of nucleic acids. A barcode-based Cas12a-mediated DNA detection strategy was introduced to overcome the limitations of current methods and enable universal detection, simultaneous detection of multiple targets, and quantitative detection. This method showed excellent performance in detecting β-thalassemia and spinal muscular atrophy patients.