Single-molecule perspectives of CRISPR/Cas systems: target search, recognition, and cleavage

CRISPR/Cas systems have emerged as powerful tools for gene editing, nucleic acid detection, and therapeutic applications. Recent advances in single-molecule techniques have provided new insights into the DNA-targeting mechanisms of CRISPR/ Cas systems, in particular, Types I, II, and V. Here, we review how single-molecule approaches have expanded our understanding of key processes, namely target search, recognition, and cleavage. Furthermore, we focus on the dynamic behavior of Cas proteins, including PAM site recognition and R-loop formation, which are crucial to ensure specificity and efficiency in gene editing. Additionally, we discuss the conformational changes and interactions that drive precise DNA cleavage by different Cas proteins. This mini review provides a comprehensive overview of CRISPR/Cas molecular dynamics, offering conclusive insights into their broader potential for genome editing and biotechnological applications. [BMB Reports 2025; 58(1): 8-16].