The trans-activating CRISPR RNA (tracrRNA) is fundamental to the CRISPR/Cas9 system, forming guide RNA with crRNA. Despite its known importance in crRNA maturation and Cas9 RNP-mediated DNA cleavage, the exact function of tracrRNA scaffolds remains unclear. In this investigation, we generated five tracrRNA variants by removing specific scaffolds, including Stem loops 1, 2, and 3, and the Linker. Using a new single-molecule assay, we directly observed target binding and cleavage processes guided by Cas9 RNP. Our findings underscore the vital role of the Linker in initiating R-loops and highlight the significance of Stem loop 2 in identifying PAM-distal mismatches within target DNA. Furthermore, we explored cleavage efficiency by adding tracrRNA segments, indicating that maintaining the integrity of Stem loops 2 and 3 is crucial for potent Cas9 activity. We believe that these results deepen our understanding of Cas9 functionality and offer insights into its detailed mechanism from target binding to cleavage.