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

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.

doi:10.1038/s41467-019-10577-3

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

Journal Article · Wed Jun 26 00:00:00 EDT 2019 · Nature Communications

DOI:https://doi.org/10.1038/s41467-019-10577-3· OSTI ID:1603516

Thavalingam, Annoj ^[1]; Cheng, Zhi ^[2]; Garcia, Bianca ^[1]; Huang, Xue ^[3]; Shah, Megha ^[1]; Sun, Wei ^[4]; Wang, Min ^[4]; Harrington, Lucas ^[5]; Hwang, Sungwon ^[1]; Hidalgo-Reyes, Yurima ^[1]; ^[6]; ^[7]; ^[1]; Moraes, Trevor F. ^[1]; Wang, Yanli ^[2]; ^[1]

Univ. of Toronto, ON (Canada)
Chinese Academy of Science (CAS), Beijing (China); Univ. of Chinese Academy of Sciences, Beijing (China)
Chinese Academy of Science (CAS), Beijing (China); Univ. of Science and Technology of China, Hefei (China)
Chinese Academy of Science (CAS), Beijing (China)
Univ. of California, Berkeley, CA (United States)
Univ. of Massachusetts, Worcester, MA (United States)
Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

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.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: 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)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1603516

Journal Information:: Nature Communications, Journal Name: Nature Communications Journal Issue: 1 Vol. 10; ISSN 2041-1723

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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Exploitation of the Cooperative Behaviors of Anti-CRISPR Phages Chevallereau, Anne; Meaden, Sean; Fradet, Olivier Cell Host & Microbe, Vol. 27, Issue 2 https://doi.org/10.1016/j.chom.2019.12.004	journal	February 2020
CRISPR-Cas Systems and the Paradox of Self-Targeting Spacers Wimmer, Franziska; Beisel, Chase L. Frontiers in Microbiology, Vol. 10 https://doi.org/10.3389/fmicb.2019.03078	journal	January 2020
Anti‐CRISPR proteins targeting the CRISPR‐Cas system enrich the toolkit for genetic engineering Liu, Qiong; Zhang, Hongxia; Huang, Xiaotian The FEBS Journal, Vol. 287, Issue 4 https://doi.org/10.1111/febs.15139	journal	November 2019

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