RNA Research Division, New England Biolabs, Inc., Beverly, MA 01915, USA.
Nucleic Acids Res. 2023 May 8;51(8):3903-3917. doi: 10.1093/nar/gkad233.
The RNA-guided Cas9 endonuclease from Staphylococcus aureus (SauCas9) can catalyze multiple-turnover reactions whereas Cas9 from Streptococcus pyogenes (SpyCas9) is a single-turnover enzyme. Here we dissect the mechanism of multiple-turnover catalysis by SauCas9 and elucidate its molecular basis. We show that the multiple-turnover catalysis does not require more than stoichiometric RNA guides to Cas9 nuclease. Rather, the RNA-guide loaded ribonucleoprotein (RNP) is the reactive unity that is slowly released from product and recycled in the subsequent reaction. The mechanism that RNP is recycled for multiple-turnover reaction entails the unwinding of the RNA:DNA duplex in the R-loop. We argue that DNA rehybridization is required for RNP release by supplementing the energy cost in the process. Indeed, turnover is arrested when DNA rehybridization is suppressed. Further, under higher salt conditions, both SauCas9 and SpyCas9 showed increased turnover, and engineered SpyCas9 nucleases that form fewer direct or hydrogen bonding interactions with target DNA became multiple-turnover enzymes. Thus, these results indicate that for both SpyCas9 and SauCas9, turnover is determined by the energetic balance of the post-chemistry RNP-DNA interaction. Due to the conserved protein core folds, the mechanism underpinning turnover we establish here is likely operant in all Cas9 nucleases.
来自金黄色葡萄球菌（SauCas9）的 RNA 指导 Cas9 内切酶可以催化多次周转反应，而来自酿脓链球菌（SpyCas9）的 Cas9 则是单次周转酶。在这里，我们剖析了 SauCas9 多次周转催化的机制，并阐明了其分子基础。我们表明，多次周转催化不需要超过 Cas9 核酸酶的化学计量 RNA 向导。相反，加载 RNA 的核糖核蛋白（RNP）是从产物中缓慢释放并在随后的反应中循环的反应性单元。RNP 循环进行多次周转反应的机制需要 R 环中 RNA:DNA 双链的解旋。我们认为，通过补充该过程中的能量成本，DNA 重新杂交是 RNP 释放所必需的。实际上，当抑制 DNA 重新杂交时，周转会被阻止。此外，在较高盐条件下，SauCas9 和 SpyCas9 的周转率均增加，并且与靶 DNA 形成较少直接或氢键相互作用的工程化 SpyCas9 核酸酶成为多次周转酶。因此，这些结果表明，对于 SpyCas9 和 SauCas9 而言，周转率取决于化学后 RNP-DNA 相互作用的能量平衡。由于保守的蛋白质核心折叠，我们在这里建立的周转率的基础机制可能在所有 Cas9 核酸酶中都起作用。
