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Its small size facilitates in\u00a0vivo delivery for genome editing in various organisms. Herein, using single\u2010molecule and ensemble approaches, we systemically study the mechanism of SaCas9 underlying its interplay with DNA. We find that the DNA binding and cleavage of SaCas9 require complementarities of 6\u2010 and 18\u2010bp of PAM\u2010proximal DNA with guide RNA, respectively. These activities are mediated by two steady interactions among the ternary complex, one of which is located approximately 6\u00a0bp from the PAM and beyond the apparent footprint of SaCas9 on DNA. Notably, the other interaction within the protospacer is significantly strong and thus poses DNA\u2010bound SaCas9 a persistent block to DNA\u2010tracking motors. Intriguingly, after cleavage, SaCas9 autonomously releases the PAM\u2010distal DNA while retaining binding to the PAM. This partial DNA release immediately abolishes its strong interaction with the protospacer DNA and consequently promotes its subsequent dissociation from the PAM. Overall, these data provide a dynamic understanding of SaCas9 and instruct its effective applications.\n                  <\/jats:p>","DOI":"10.15252\/embr.202050184","type":"journal-article","created":{"date-parts":[[2020,8,13]],"date-time":"2020-08-13T09:35:14Z","timestamp":1597311314000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":35,"title":["Dynamics of Staphylococcus aureus Cas9 in DNA target Association and Dissociation"],"prefix":"10.1038","volume":"21","author":[{"given":"Siqi","family":"Zhang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9187-4277","authenticated-orcid":false,"given":"Qian","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xi\u2010Miao","family":"Hou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lijuan","family":"Guo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fangzhu","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2651-0784","authenticated-orcid":false,"given":"Lulu","family":"Bi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xia","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hai\u2010Hong","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fengcai","family":"Wen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xu\u2010Guang","family":"Xi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xingxu","family":"Huang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bin","family":"Shen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4590-7795","authenticated-orcid":false,"given":"Bo","family":"Sun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2020,8,13]]},"reference":[{"key":"BFEMBR202050184_CR1","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1038\/nature24268","volume":"550","author":"JS Chen","year":"2017","unstructured":"Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA (2017) Enhanced proofreading governs CRISPR\u2010Cas9 targeting accuracy. 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