Critical role of Cas9 aggregation on in vitro DNA cleavage

The CRISPR/Cas9 system is a powerful genome-editing tool widely used in molecular biology and gene therapy, whose efficiency strongly depends on the physicochemical properties of the Cas9 ribonucleoprotein complex. Optimizing Cas9 activity remains essential for reliable genome-editing applications, yet the factors limiting its in vitro cleavage efficiency are not fully understood. Among these, protein aggregation has been suggested to critically impair Cas9 functionality, although its role has not been systematically analysed. Here, we investigate Cas9 aggregation under different environmental conditions and evaluate its impact on in vitro DNA cleavage efficiency. Using fluorescently labelled Cas9 and single-molecule fluorescence techniques, we quantify aggregation as a function of buffer composition, ionic strength, salt concentration and sgRNA presence, and relate these properties to cleavage activity. Our results show that Cas9 aggregation significantly reduces DNA cleavage efficiency, with higher aggregation levels consistently correlating with lower activity. In contrast, buffers with higher ionic strength or stabilizing components reduce aggregation and enhance Cas9 performance. Overall, this study demonstrates that Cas9 aggregation plays a critical role in determining in vitro cleavage efficiency and highlights the importance of controlling protein aggregation to optimize CRISPR/Cas9-based genome-editing applications and delivery strategies.