Fluorescence Detection of Low-Abundant Nucleic Acids by Spatially Confined Cas14a1 Reactions

ABSTRACT

Sensitive and specific diagnosis of diseases is necessary for effective identification and treatment; however accurate diagnosis remains a challenge. The CRISPR-Cas system is a nucleic acid-based adaptive immune system with enzymatic activity discovered in archaea and bacteria that protects them from viral infection. Due to their selectivity and programmability, CRISPR-Cas systems have been involved in the development of biosensors and biosensing systems for the detection of nucleic acids. In this study, we attempt to develop a low-abundant DNA detection assay based on Cas14a1 proteins by spatial confinement in 2D and 3D models. Purification and functional characterization of Cas14a1- RNA complex were performed. Both 2D and 3D assays detect a target DNA by continuous endonuclease reactions that release a quencher from a fluorescent reporter. The confinement in 2D was accomplished by tethering Cas14a1 proteins to planar membrane microarrays, while the 3D model used polydisperse droplets to confine Cas14a1 in microscopic volumes. On 2D membranes, signal was limited by reporter/tether geometry and off-site cleavage; in 3D droplets, detection was feasible but ultimately constrained by the enzyme’s slow turnover. Spatial confinement remains a useful physical principle for target DNA amplification-free assays. Confinement-based assays could be further improved to deliver faster and more sensitive detection by optimizing tethering/reporters designs and employing faster Cas enzyme variants.