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

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

Journal Article · 26 June 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.

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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 (NSFC); Chinese Academy of Sciences (CAS)

Grant/Contract Number:: AC02-05CH11231; RGPIN-2018-06546; GM125797; 2017YFA0504203; 31725008; 31630015; 31571335; 31700662; QYZDY-SSW-SMC021

OSTI ID:: 1603516

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

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 41 works

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References (60)

Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage Jiang, F.; Taylor, D. W.; Chen, J. S. Science, Vol. 351, Issue 6275 https://doi.org/10.1126/science.aad8282	journal	January 2016
Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein Pawluk, April; Shah, Megha; Mejdani, Marios mBio, Vol. 8, Issue 6 https://doi.org/10.1128/mBio.01751-17	journal	December 2017
Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation Jinek, M.; Jiang, F.; Taylor, D. W. Science, Vol. 343, Issue 6176 https://doi.org/10.1126/science.1247997	journal	February 2014
An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9 Hynes, Alexander P.; Rousseau, Geneviève M.; Lemay, Marie-Laurence Nature Microbiology, Vol. 2, Issue 10 https://doi.org/10.1038/s41564-017-0004-7	journal	August 2017
Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system Bondy-Denomy, Joe; Pawluk, April; Maxwell, Karen L. Nature, Vol. 493, Issue 7432 https://doi.org/10.1038/nature11723	journal	December 2012
Structure Reveals Mechanisms of Viral Suppressors that Intercept a CRISPR RNA-Guided Surveillance Complex Chowdhury, Saikat; Carter, Joshua; Rollins, MaryClare F. Cell, Vol. 169, Issue 1 https://doi.org/10.1016/j.cell.2017.03.012	journal	March 2017
Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA Nishimasu, Hiroshi; Ran, F. Ann; Hsu, Patrick D. Cell, Vol. 156, Issue 5 https://doi.org/10.1016/j.cell.2014.02.001	journal	February 2014
A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity Jinek, M.; Chylinski, K.; Fonfara, I. Science, Vol. 337, Issue 6096, p. 816-821 https://doi.org/10.1126/science.1225829	journal	June 2012
Dali server: conservation mapping in 3D Holm, Liisa; Rosenstrï¿½m, Pï¿½ivi Nucleic Acids Research, Vol. 38, Issue suppl_2 https://doi.org/10.1093/nar/gkq366	journal	May 2010
Systematic discovery of natural CRISPR-Cas12a inhibitors Watters, Kyle E.; Fellmann, Christof; Bai, Hua B. Science, Vol. 362, Issue 6411 https://doi.org/10.1126/science.aau5138	journal	September 2018
Crystal Structure of Staphylococcus aureus Cas9 Nishimasu, Hiroshi; Cong, Le; Yan, Winston X. Cell, Vol. 162, Issue 5 https://doi.org/10.1016/j.cell.2015.08.007	journal	August 2015
A New Group of Phage Anti-CRISPR Genes Inhibits the Type I-E CRISPR-Cas System of Pseudomonas aeruginosa Pawluk, April; Bondy-Denomy, Joseph; Cheung, Vivian H. W. mBio, Vol. 5, Issue 2 https://doi.org/10.1128/mBio.00896-14	journal	April 2014
Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species Pawluk, April; Staals, Raymond H. J.; Taylor, Corinda Nature Microbiology, Vol. 1, Issue 8 https://doi.org/10.1038/nmicrobiol.2016.85	journal	June 2016
Naturally Occurring Off-Switches for CRISPR-Cas9 Pawluk, April; Amrani, Nadia; Zhang, Yan Cell, Vol. 167, Issue 7 https://doi.org/10.1016/j.cell.2016.11.017	journal	December 2016
Multiple mechanisms for CRISPR–Cas inhibition by anti-CRISPR proteins Bondy-Denomy, Joseph; Garcia, Bianca; Strum, Scott Nature, Vol. 526, Issue 7571 https://doi.org/10.1038/nature15254	journal	September 2015
T7 Vectors with a Modified T7lacPromoter for Expression of Proteins inEscherichia coli Peränen, Johan; Rikkonen, Marja; Hyvönen, Marko Analytical Biochemistry, Vol. 236, Issue 2 https://doi.org/10.1006/abio.1996.0187	journal	May 1996
A Broad-Spectrum Inhibitor of CRISPR-Cas9 Harrington, Lucas B.; Doxzen, Kevin W.; Ma, Enbo Cell, Vol. 170, Issue 6 https://doi.org/10.1016/j.cell.2017.07.037	journal	September 2017
Anti-CRISPR proteins encoded by archaeal lytic viruses inhibit subtype I-D immunity He, Fei; Bhoobalan-Chitty, Yuvaraj; Van, Lan B. Nature Microbiology, Vol. 3, Issue 4 https://doi.org/10.1038/s41564-018-0120-z	journal	March 2018
Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes Brouns, S. J. J.; Jore, M. M.; Lundgren, M. Science, Vol. 321, Issue 5891, p. 960-964 https://doi.org/10.1126/science.1159689	journal	August 2008
Diverse Mechanisms of CRISPR-Cas9 Inhibition by Type IIC Anti-CRISPR Proteins Zhu, Yalan; Gao, Ang; Zhan, Qi Molecular Cell, Vol. 74, Issue 2 https://doi.org/10.1016/j.molcel.2019.01.038	journal	April 2019
Cas9 function and host genome sampling in Type II-A CRISPR–Cas adaptation Wei, Yunzhou; Terns, Rebecca M.; Terns, Michael P. Genes & Development, Vol. 29, Issue 4 https://doi.org/10.1101/gad.257550.114	journal	February 2015
Structural basis of CRISPR–SpyCas9 inhibition by an anti-CRISPR protein Dong, De; Guo, Minghui; Wang, Sihan Nature, Vol. 546, Issue 7658 https://doi.org/10.1038/nature22377	journal	April 2017
Annotation and Classification of CRISPR-Cas Systems Makarova, Kira S.; Koonin, Eugene V. CRISPR, p. 47-75 https://doi.org/10.1007/978-1-4939-2687-9_4	book	January 2015
Diversity, classification and evolution of CRISPR-Cas systems Koonin, Eugene V.; Makarova, Kira S.; Zhang, Feng Current Opinion in Microbiology, Vol. 37 https://doi.org/10.1016/j.mib.2017.05.008	journal	June 2017
Phage-Encoded Anti-CRISPR Defenses Stanley, Sabrina Y.; Maxwell, Karen L. Annual Review of Genetics, Vol. 52, Issue 1 https://doi.org/10.1146/annurev-genet-120417-031321	journal	November 2018
Bacteriophage Cooperation Suppresses CRISPR-Cas3 and Cas9 Immunity Borges, Adair L.; Zhang, Jenny Y.; Rollins, MaryClare F. Cell, Vol. 174, Issue 4 https://doi.org/10.1016/j.cell.2018.06.013	journal	August 2018
Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex Guo, Tai Wei; Bartesaghi, Alberto; Yang, Hui Cell, Vol. 171, Issue 2 https://doi.org/10.1016/j.cell.2017.09.006	journal	October 2017
Inhibition of CRISPR-Cas9 with Bacteriophage Proteins Rauch, Benjamin J.; Silvis, Melanie R.; Hultquist, Judd F. Cell, Vol. 168, Issue 1-2 https://doi.org/10.1016/j.cell.2016.12.009	journal	January 2017
Discovery of widespread type I and type V CRISPR-Cas inhibitors Marino, Nicole D.; Zhang, Jenny Y.; Borges, Adair L. Science, Vol. 362, Issue 6411 https://doi.org/10.1126/science.aau5174	journal	September 2018
Structural basis of Cas3 inhibition by the bacteriophage protein AcrF3 Wang, Xiaofei; Yao, Deqiang; Xu, Jin-Gen Nature Structural & Molecular Biology, Vol. 23, Issue 9 https://doi.org/10.1038/nsmb.3269	journal	July 2016
A Cas9-guide RNA complex preorganized for target DNA recognition Jiang, F.; Zhou, K.; Ma, L. Science, Vol. 348, Issue 6242 https://doi.org/10.1126/science.aab1452	journal	June 2015
CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes Barrangou, R.; Fremaux, C.; Deveau, H. Science, Vol. 315, Issue 5819 https://doi.org/10.1126/science.1138140	journal	March 2007
RNA-Independent DNA Cleavage Activities of Cas9 and Cas12a Sundaresan, Ramya; Parameshwaran, Hari Priya; Yogesha, S. D. Cell Reports, Vol. 21, Issue 13 https://doi.org/10.1016/j.celrep.2017.11.100	journal	December 2017
Cas9 specifies functional viral targets during CRISPR–Cas adaptation Heler, Robert; Samai, Poulami; Modell, Joshua W. Nature, Vol. 519, Issue 7542 https://doi.org/10.1038/nature14245	journal	February 2015
Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins Hynes, Alexander P.; Rousseau, Geneviève M.; Agudelo, Daniel Nature Communications, Vol. 9, Issue 1 https://doi.org/10.1038/s41467-018-05092-w	journal	July 2018
CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA Marraffini, Luciano A.; Sontheimer, Erik J. Science, Vol. 322, Issue 5909 https://doi.org/10.1126/science.1165771	journal	December 2008
Anti-CRISPR Phages Cooperate to Overcome CRISPR-Cas Immunity Landsberger, Mariann; Gandon, Sylvain; Meaden, Sean Cell, Vol. 174, Issue 4 https://doi.org/10.1016/j.cell.2018.05.058	journal	August 2018
Alternate binding modes of anti-CRISPR viral suppressors AcrF1/2 to Csy surveillance complex revealed by cryo-EM structures Peng, Ruchao; Xu, Ying; Zhu, Tengfei Cell Research, Vol. 27, Issue 7 https://doi.org/10.1038/cr.2017.79	journal	June 2017
Structures of Cas9 endonucleases reveal RNA-mediated conformational activation Jinek, Martin; Jiang, Fuguo; Taylor, David W. American Association for the Advancement of Science https://doi.org/10.5167/uzh-96990	text	January 2014
T7 Vectors with a Modified T7lacPromoter for Expression of Proteins inEscherichia coli Peränen, Johan; Rikkonen, Marja; Hyvönen, Marko Analytical Biochemistry, Vol. 236, Issue 2 https://doi.org/10.1006/abio.1996.0187	journal	May 1996
Annotation and Classification of CRISPR-Cas Systems Makarova, Kira S.; Koonin, Eugene V. CRISPR, p. 47-75 https://doi.org/10.1007/978-1-4939-2687-9_4	book	January 2015
Naturally Occurring Off-Switches for CRISPR-Cas9 Pawluk, April; Amrani, Nadia; Zhang, Yan Cell, Vol. 167, Issue 7 https://doi.org/10.1016/j.cell.2016.11.017	journal	December 2016
Structure Reveals Mechanisms of Viral Suppressors that Intercept a CRISPR RNA-Guided Surveillance Complex Chowdhury, Saikat; Carter, Joshua; Rollins, MaryClare F. Cell, Vol. 169, Issue 1 https://doi.org/10.1016/j.cell.2017.03.012	journal	March 2017
Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex Guo, Tai Wei; Bartesaghi, Alberto; Yang, Hui Cell, Vol. 171, Issue 2 https://doi.org/10.1016/j.cell.2017.09.006	journal	October 2017
Anti-CRISPR Phages Cooperate to Overcome CRISPR-Cas Immunity Landsberger, Mariann; Gandon, Sylvain; Meaden, Sean Cell, Vol. 174, Issue 4 https://doi.org/10.1016/j.cell.2018.05.058	journal	August 2018
Bacteriophage Cooperation Suppresses CRISPR-Cas3 and Cas9 Immunity Borges, Adair L.; Zhang, Jenny Y.; Rollins, MaryClare F. Cell, Vol. 174, Issue 4 https://doi.org/10.1016/j.cell.2018.06.013	journal	August 2018
Alternate binding modes of anti-CRISPR viral suppressors AcrF1/2 to Csy surveillance complex revealed by cryo-EM structures Peng, Ruchao; Xu, Ying; Zhu, Tengfei Cell Research, Vol. 27, Issue 7 https://doi.org/10.1038/cr.2017.79	journal	June 2017
Cas9 specifies functional viral targets during CRISPR–Cas adaptation Heler, Robert; Samai, Poulami; Modell, Joshua W. Nature, Vol. 519, Issue 7542 https://doi.org/10.1038/nature14245	journal	February 2015
Multiple mechanisms for CRISPR–Cas inhibition by anti-CRISPR proteins Bondy-Denomy, Joseph; Garcia, Bianca; Strum, Scott Nature, Vol. 526, Issue 7571 https://doi.org/10.1038/nature15254	journal	September 2015
Structural basis of CRISPR–SpyCas9 inhibition by an anti-CRISPR protein Dong, De; Guo, Minghui; Wang, Sihan Nature, Vol. 546, Issue 7658 https://doi.org/10.1038/nature22377	journal	April 2017
Structural basis of Cas3 inhibition by the bacteriophage protein AcrF3 Wang, Xiaofei; Yao, Deqiang; Xu, Jin-Gen Nature Structural & Molecular Biology, Vol. 23, Issue 9 https://doi.org/10.1038/nsmb.3269	journal	July 2016
Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins Hynes, Alexander P.; Rousseau, Geneviève M.; Agudelo, Daniel Nature Communications, Vol. 9, Issue 1 https://doi.org/10.1038/s41467-018-05092-w	journal	July 2018
Anti-CRISPR proteins encoded by archaeal lytic viruses inhibit subtype I-D immunity He, Fei; Bhoobalan-Chitty, Yuvaraj; Van, Lan B. Nature Microbiology, Vol. 3, Issue 4 https://doi.org/10.1038/s41564-018-0120-z	journal	March 2018
Dali server: conservation mapping in 3D Holm, Liisa; Rosenstrï¿½m, Pï¿½ivi Nucleic Acids Research, Vol. 38, Issue suppl_2 https://doi.org/10.1093/nar/gkq366	journal	May 2010
Guide RNA engineering for versatile Cas9 functionality Nowak, Chance M.; Lawson, Seth; Zerez, Megan Nucleic Acids Research https://doi.org/10.1093/nar/gkw908	journal	October 2016
CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA Marraffini, Luciano A.; Sontheimer, Erik J. Science, Vol. 322, Issue 5909 https://doi.org/10.1126/science.1165771	journal	December 2008
Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation Jinek, M.; Jiang, F.; Taylor, D. W. Science, Vol. 343, Issue 6176 https://doi.org/10.1126/science.1247997	journal	February 2014
Systematic discovery of natural CRISPR-Cas12a inhibitors Watters, Kyle E.; Fellmann, Christof; Bai, Hua B. Science, Vol. 362, Issue 6411 https://doi.org/10.1126/science.aau5138	journal	September 2018
Discovery of widespread type I and type V CRISPR-Cas inhibitors Marino, Nicole D.; Zhang, Jenny Y.; Borges, Adair L. Science, Vol. 362, Issue 6411 https://doi.org/10.1126/science.aau5174	journal	September 2018
Phage-Encoded Anti-CRISPR Defenses Stanley, Sabrina Y.; Maxwell, Karen L. Annual Review of Genetics, Vol. 52, Issue 1 https://doi.org/10.1146/annurev-genet-120417-031321	journal	November 2018

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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
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

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59 BASIC BIOLOGICAL SCIENCES
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Title: Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2

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