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

Annoj Thavalingam, Zhi Cheng, Bianca Garcia, Xue Huang, Megha Shah, Wei Sun, Min Wang, Lucas Harrington, Sungwon Hwang, Yurima Hidalgo-Reyes, Erik J. Sontheimer, Jennifer Doudna, Alan R. Davidson, Trevor F. Moraes, Yanli Wang () and Karen L. Maxwell ()
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Annoj Thavalingam: University of Toronto
Zhi Cheng: Chinese Academy of Sciences
Bianca Garcia: University of Toronto
Xue Huang: Chinese Academy of Sciences
Megha Shah: University of Toronto
Wei Sun: Chinese Academy of Sciences
Min Wang: Chinese Academy of Sciences
Lucas Harrington: University of California, Berkeley
Sungwon Hwang: University of Toronto
Yurima Hidalgo-Reyes: University of Toronto
Erik J. Sontheimer: University of Massachusetts Medical School
Jennifer Doudna: University of California, Berkeley
Alan R. Davidson: University of Toronto
Trevor F. Moraes: University of Toronto
Yanli Wang: Chinese Academy of Sciences
Karen L. Maxwell: University of Toronto

Nature Communications, 2019, vol. 10, issue 1, 1-11

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41467-019-10577-3

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