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Nat Commun. 2019 Jun 26;10(1):2806. doi: 10.1038/s41467-019-10577-3.

Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2.

Author information

1
Department of Biochemistry, University of Toronto, 661 University Avenue, Suite 1600, Toronto, ON, M5G 1M1, Canada.
2
Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
3
University of Chinese Academy of Sciences, Beijing, 100049, China.
4
Department of Molecular Genetics, University of Toronto, 661 University Avenue, Suite 1600, Toronto, ON, M5G 1M1, Canada.
5
Hefei National Research Center for Physical Sciences at the Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, 230027, Anhui, China.
6
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, 94720, USA.
7
RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, 01605, USA.
8
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, 01605, USA.
9
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.
10
Innovative Genomics Institute, University of California, Berkeley, CA, 94704, USA.
11
Howard Hughes Medical Institute, University of California, Berkeley, CA, 94720, USA.
12
Department of Chemistry, University of California, Berkeley, CA, 94720, USA.
13
Gladstone Institutes, San Francisco, CA, 94158, USA.
14
Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China. ylwang@ibp.ac.cn.
15
University of Chinese Academy of Sciences, Beijing, 100049, China. ylwang@ibp.ac.cn.
16
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China. ylwang@ibp.ac.cn.
17
Department of Biochemistry, University of Toronto, 661 University Avenue, Suite 1600, Toronto, ON, M5G 1M1, Canada. karen.maxwell@utoronto.ca.

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 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.

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