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Title: Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2

Abstract

CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements. The CRISPR-Cas9 system has been widely adopted as a powerful genome-editing tool, and phage-encoded inhibitors, known as anti-CRISPRs, offer a means of regulating its activity. Here, we report the crystal structures of anti-CRISPR protein AcrIIC2 Nme alone and in complex with Nme1Cas9. We demonstrate that AcrIIC2 Nme inhibits Cas9 through interactions with the positively charged bridge helix, thereby preventing sgRNA loading. In vivo phage plaque assays and in vitro DNA cleavage assays show that AcrIIC2 Nme mediates its activity through a large electronegative surface. This work shows that anti-CRISPR activity can be mediated through the inhibition of Cas9 complex assembly.

Authors:
 [1];  [2];  [1];  [3];  [1];  [4];  [4];  [5];  [1];  [1]; ORCiD logo [6]; ORCiD logo [7]; ORCiD logo [1];  [1];  [2]; ORCiD logo [1]
  1. Univ. of Toronto, ON (Canada)
  2. Chinese Academy of Science (CAS), Beijing (China); Univ. of Chinese Academy of Sciences, Beijing (China)
  3. Chinese Academy of Science (CAS), Beijing (China); Univ. of Science and Technology of China, Hefei (China)
  4. Chinese Academy of Science (CAS), Beijing (China)
  5. Univ. of California, Berkeley, CA (United States)
  6. Univ. of Massachusetts, Worcester, MA (United States)
  7. Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC); National Sciences and Engineering Research Council of Canada; National Institutes of Health (NIH); Chinese Ministry of Science and Technology; National Natural Science Foundation of China (NNSFC); Chinese Academy of Sciences (CAS)
OSTI Identifier:
1603516
Grant/Contract Number:  
[AC02-05CH11231; RGPIN-2018-06546; GM125797; 2017YFA0504203; 31725008; 31630015; 31571335; 31700662; QYZDY-SSW-SMC021]
Resource Type:
Accepted Manuscript
Journal Name:
Nature Communications
Additional Journal Information:
[ Journal Volume: 10; Journal Issue: 1]; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; bacteriophages; CRISPR-Cas systems; x-ray crystallography

Citation Formats

Thavalingam, Annoj, Cheng, Zhi, Garcia, Bianca, Huang, Xue, Shah, Megha, Sun, Wei, Wang, Min, Harrington, Lucas, Hwang, Sungwon, Hidalgo-Reyes, Yurima, Sontheimer, Erik J., Doudna, Jennifer, Davidson, Alan R., Moraes, Trevor F., Wang, Yanli, and Maxwell, Karen L. Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2. United States: N. p., 2019. Web. doi:10.1038/s41467-019-10577-3.
Thavalingam, Annoj, Cheng, Zhi, Garcia, Bianca, Huang, Xue, Shah, Megha, Sun, Wei, Wang, Min, Harrington, Lucas, Hwang, Sungwon, Hidalgo-Reyes, Yurima, Sontheimer, Erik J., Doudna, Jennifer, Davidson, Alan R., Moraes, Trevor F., Wang, Yanli, & Maxwell, Karen L. Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2. United States. doi:10.1038/s41467-019-10577-3.
Thavalingam, Annoj, Cheng, Zhi, Garcia, Bianca, Huang, Xue, Shah, Megha, Sun, Wei, Wang, Min, Harrington, Lucas, Hwang, Sungwon, Hidalgo-Reyes, Yurima, Sontheimer, Erik J., Doudna, Jennifer, Davidson, Alan R., Moraes, Trevor F., Wang, Yanli, and Maxwell, Karen L. Wed . "Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2". United States. doi:10.1038/s41467-019-10577-3. https://www.osti.gov/servlets/purl/1603516.
@article{osti_1603516,
title = {Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2},
author = {Thavalingam, Annoj and Cheng, Zhi and Garcia, Bianca and Huang, Xue and Shah, Megha and Sun, Wei and Wang, Min and Harrington, Lucas and Hwang, Sungwon and Hidalgo-Reyes, Yurima and Sontheimer, Erik J. and Doudna, Jennifer and Davidson, Alan R. and Moraes, Trevor F. and Wang, Yanli and Maxwell, Karen L.},
abstractNote = {CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements. The CRISPR-Cas9 system has been widely adopted as a powerful genome-editing tool, and phage-encoded inhibitors, known as anti-CRISPRs, offer a means of regulating its activity. Here, we report the crystal structures of anti-CRISPR protein AcrIIC2Nme alone and in complex with Nme1Cas9. We demonstrate that AcrIIC2Nme inhibits Cas9 through interactions with the positively charged bridge helix, thereby preventing sgRNA loading. In vivo phage plaque assays and in vitro DNA cleavage assays show that AcrIIC2Nme mediates its activity through a large electronegative surface. This work shows that anti-CRISPR activity can be mediated through the inhibition of Cas9 complex assembly.},
doi = {10.1038/s41467-019-10577-3},
journal = {Nature Communications},
number = [1],
volume = [10],
place = {United States},
year = {2019},
month = {6}
}

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