Nucleic Acid Enzyme‐Activated CRISPR‐Cas12a With Circular CRISPR RNA for Biosensing

Abstractclustered regularly interspaced short palindromic repeats (CRISPR)‐Cas systems are increasingly used in biosensor development. However, directly translating recognition events for non‐nucleic acid targets by CRISPR into effective measurable signals represents an important ongoing challenge. Herein, it is hypothesized and confirmed that CRISPR RNAs (crRNAs) in a circular topology efficiently render Cas12a incapable of both site‐specific double‐stranded DNA cutting and nonspecific single‐stranded DNA trans cleavage. Importantly, it is shown that nucleic acid enzymes (NAzymes) with RNA‐cleaving activity can linearize the circular crRNAs, activating CRISPR‐Cas12a functions. Using ligand‐responsive ribozymes and DNAzymes as molecular recognition elements, it is demonstrated that target‐triggered linearization of circular crRNAs offers great versatility for biosensing. This strategy is termed as “NAzyme‐Activated CRISPR‐Cas12a with Circular CRISPR RNA (NA3C).” Use of NA3C for clinical evaluation of urinary tract infections using an Escherichia coli‐responsive RNA‐cleaving DNAzyme to test 40 patient urine samples, providing a diagnostic sensitivity of 100% and specificity of 90%, is further demonstrated.

Nucleic Acid Enzyme‐Activated CRISPR‐Cas12a With Circular CRISPR RNA for Biosensing Journal Articles