Toward point-of-care CRISPR-based diagnostics using split crRNA and split activator/target systems.

Abstract

Split crRNA and split activator/target strategies have enhanced and advanced CRISPR-based diagnostics (CRISPR/Dx) by requiring a conditional assembly of components. The split crRNA involves dividing the guide RNA (crRNA) itself into multiple inactive fragments that only reassemble to become functional crRNA upon binding to a specific target. The split activator/target involves dividing the target or activator substrate into two reconfigurable segments that trigger Cas activation only upon proper assembly. These evolved CRISPR/Dx systems have enabled: direct quantification of miRNA and ultrashort RNA sequences, amplification-free and attomolar-level detection, single-base discrimination, protospacer adjacent motif (PAM)-free recognition, one-pot analysis, multiplex diagnostics, non-nucleic acid target detection, and constructing biochemical circuits and logic gates. Herein, we critically discuss the advances, pitfalls, challenges, and future perspectives of these CRISPR/Dx systems for field-deployable point-of-care diagnostics. Different split crRNA and split activator/target strategies and applications for CRISPR/Dx are highlighted.