Shunhang Liu, Xichen Rao, Ruiliang Zhao, Wenyuan Han
{"title":"Cas12a的反式DNA裂解活性对质粒或噬菌体没有可检测到的免疫力。","authors":"Shunhang Liu, Xichen Rao, Ruiliang Zhao, Wenyuan Han","doi":"10.3389/fgeed.2022.929929","DOIUrl":null,"url":null,"abstract":"<p><p>Cas12a is a type V-A CRISPR-Cas RNA-guided endonuclease. It cleaves dsDNA at specific site, and then is activated for nonspecific ssDNA cleavage in trans <i>in vitro</i>. The immune function of the trans activity is still unknown. To address this question, we constructed a Cas12a targeting system in <i>Escherichia coli</i>, where Cas12a cleaved a high-copy target plasmid to unleash the trans ssDNA cleavage activity. Then, we analyzed the effect of the Cas12a targeting on a non-target plasmid and a ssDNA phage. The results show that Cas12a efficiently eliminates target plasmid but exerts no impact on the maintenance of the non-target plasmid or plague formation efficiency of the phage. In addition, a two-spacer CRISPR array, which facilitates target plasmid depletion, still has no detectable effect on the non-target plasmid or phage either. Together, the data suggest that the trans ssDNA cleavage of Cas12a does not contribute to immunity <i>in vivo</i>.</p>","PeriodicalId":4,"journal":{"name":"ACS Applied Energy Materials","volume":" ","pages":"929929"},"PeriodicalIF":5.4000,"publicationDate":"2022-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9360544/pdf/","citationCount":"3","resultStr":"{\"title\":\"The trans DNA cleavage activity of Cas12a provides no detectable immunity against plasmid or phage.\",\"authors\":\"Shunhang Liu, Xichen Rao, Ruiliang Zhao, Wenyuan Han\",\"doi\":\"10.3389/fgeed.2022.929929\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Cas12a is a type V-A CRISPR-Cas RNA-guided endonuclease. It cleaves dsDNA at specific site, and then is activated for nonspecific ssDNA cleavage in trans <i>in vitro</i>. The immune function of the trans activity is still unknown. To address this question, we constructed a Cas12a targeting system in <i>Escherichia coli</i>, where Cas12a cleaved a high-copy target plasmid to unleash the trans ssDNA cleavage activity. Then, we analyzed the effect of the Cas12a targeting on a non-target plasmid and a ssDNA phage. The results show that Cas12a efficiently eliminates target plasmid but exerts no impact on the maintenance of the non-target plasmid or plague formation efficiency of the phage. In addition, a two-spacer CRISPR array, which facilitates target plasmid depletion, still has no detectable effect on the non-target plasmid or phage either. Together, the data suggest that the trans ssDNA cleavage of Cas12a does not contribute to immunity <i>in vivo</i>.</p>\",\"PeriodicalId\":4,\"journal\":{\"name\":\"ACS Applied Energy Materials\",\"volume\":\" \",\"pages\":\"929929\"},\"PeriodicalIF\":5.4000,\"publicationDate\":\"2022-07-26\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9360544/pdf/\",\"citationCount\":\"3\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"ACS Applied Energy Materials\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.3389/fgeed.2022.929929\",\"RegionNum\":3,\"RegionCategory\":\"材料科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2022/1/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"Q2\",\"JCRName\":\"CHEMISTRY, PHYSICAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Applied Energy Materials","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.3389/fgeed.2022.929929","RegionNum":3,"RegionCategory":"材料科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2022/1/1 0:00:00","PubModel":"eCollection","JCR":"Q2","JCRName":"CHEMISTRY, PHYSICAL","Score":null,"Total":0}
The trans DNA cleavage activity of Cas12a provides no detectable immunity against plasmid or phage.
Cas12a is a type V-A CRISPR-Cas RNA-guided endonuclease. It cleaves dsDNA at specific site, and then is activated for nonspecific ssDNA cleavage in trans in vitro. The immune function of the trans activity is still unknown. To address this question, we constructed a Cas12a targeting system in Escherichia coli, where Cas12a cleaved a high-copy target plasmid to unleash the trans ssDNA cleavage activity. Then, we analyzed the effect of the Cas12a targeting on a non-target plasmid and a ssDNA phage. The results show that Cas12a efficiently eliminates target plasmid but exerts no impact on the maintenance of the non-target plasmid or plague formation efficiency of the phage. In addition, a two-spacer CRISPR array, which facilitates target plasmid depletion, still has no detectable effect on the non-target plasmid or phage either. Together, the data suggest that the trans ssDNA cleavage of Cas12a does not contribute to immunity in vivo.
期刊介绍:
ACS Applied Energy Materials is an interdisciplinary journal publishing original research covering all aspects of materials, engineering, chemistry, physics and biology relevant to energy conversion and storage. The journal is devoted to reports of new and original experimental and theoretical research of an applied nature that integrate knowledge in the areas of materials, engineering, physics, bioscience, and chemistry into important energy applications.