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

Abstract

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].

Fig. 1

DNA-targeting CRISPR/Cas systems. DNA-targeting CRISPR/Cas systems operate in three stages, based on distinct targeting mechanisms. (A) Type I involves Cascade, which identifies PAM and recruits Cas3 for DNA nicking along a single strand. (B) In Type II, Cas9 pairs with crRNA, locates PAM, and induces blunt dsDNA breaks by aligning its HNH and RuvC domains. (C) Type V uses Cas12 with a crRNA guide to recognize PAM, cleaving the non-target strand first, followed by the target strand through the RuvC domain, which after a staggered dsDNA break is formed, remains active for further ssDNA cleavage.