Structural and Mechanistic Investigations of a High-Fidelity Cas9 from <i>Parasutterella secunda</i>

The RNA-guided CRISPR-Cas9 system has transformed the field of genome editing, offering unparalleled programmability and efficiency in a wide range of applications. Type II-A Cas9 from Streptococcus pyogenes (SpCas9) is the most widely utilized system due to its robust activity. However, the high off-target effects of SpCas9 pose significant safety concerns, particularly in clinical and therapeutic settings. In this study, we report the high-resolution cryo-electron microscopy structures of Parasutterella secunda Cas9 (PsCas9), a type II-B Cas9 with substantially lower off-target effects compared to SpCas9. Both the binary complex containing sgRNA and the ternary complex containing sgRNA and target double-stranded DNA are resolved. Our analyses reveal key molecular mechanisms underlying the function of type II-B Cas9 and provide insights into the high fidelity of PsCas9.