Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a

Knott, Gavin J.; Cress, Brady F.; Liu, Jun-Jie; Thornton, Brittney W.; Lew, Rachel J.; Al-Shayeb, Basem; Rosenberg, Daniel J.; Hammel, Michal; Adler, Benjamin A.; Lobba, Marco J.; Xu, Michael; Arkin, Adam P.; Fellmann, Christof; Doudna, Jennifer A.

doi:10.7554/eLife.49110

Title: Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a

Journal Article · Fri Aug 09 00:00:00 EDT 2019 · eLife

DOI:https://doi.org/10.7554/eLife.49110· OSTI ID:1560609

^[1]; Cress, Brady F. ^[1]; Liu, Jun-Jie ^[1]; Thornton, Brittney W. ^[1]; Lew, Rachel J. ^[2]; Al-Shayeb, Basem ^[1];

^[3]; Hammel, Michal ^[4]; Adler, Benjamin A. ^[1]; Lobba, Marco J. ^[1]; Xu, Michael ^[1];

^[5];

^[6];

^[7]

Univ. of California, Berkeley, CA (United States)
Gladstone Inst., San Francisco, CA (United States)
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Gladstone Inst., San Francisco, CA (United States); Univ. of California, San Francisco, CA (United States)
Univ. of California, Berkeley, CA (United States); Gladstone Inst., San Francisco, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the L. bacterium Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The Ac. species Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.

View Accepted Manuscript (DOE)

Cite

Export

Save

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

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1560609

Journal Information:: eLife, Vol. 8; ISSN 2050-084X

Publisher:: eLife Sciences Publications, Ltd.Copyright Statement

Country of Publication:: United States

Language:: English

Citation Metrics:

Cited by: 34 works

Citation information provided by
Web of Science

References (65)

Bacteriophage resistance mechanisms Labrie, Simon J.; Samson, Julie E.; Moineau, Sylvain Nature Reviews Microbiology, Vol. 8, Issue 5 https://doi.org/10.1038/nrmicro2315	journal	March 2010
Structural Basis for the Inhibition of CRISPR-Cas12a by Anti-CRISPR Proteins Zhang, Heng; Li, Zhuang; Daczkowski, Courtney M. Cell Host & Microbe, Vol. 25, Issue 6 https://doi.org/10.1016/j.chom.2019.05.004	journal	June 2019
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
Modeling of loops in protein structures Fiser, András; Do, Richard Kinh Gian; Šali, Andrej Protein Science, Vol. 9, Issue 9 https://doi.org/10.1110/ps.9.9.1753	journal	January 2000
Csy4 is responsible for CRISPR RNA processing in Pectobacterium atrosepticum Przybilski, Rita; Richter, Corinna; Gristwood, Tamzin RNA Biology, Vol. 8, Issue 3 https://doi.org/10.4161/rna.8.3.15190	journal	May 2011
Meet the Anti-CRISPRs: Widespread Protein Inhibitors of CRISPR-Cas Systems Hwang, Sungwon; Maxwell, Karen L. The CRISPR Journal, Vol. 2, Issue 1 https://doi.org/10.1089/crispr.2018.0052	journal	February 2019
BioXTAS RAW : improvements to a free open-source program for small-angle X-ray scattering data reduction and analysis Hopkins, Jesse Bennett; Gillilan, Richard E.; Skou, Soren Journal of Applied Crystallography, Vol. 50, Issue 5 https://doi.org/10.1107/S1600576717011438	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
Functionally diverse type V CRISPR-Cas systems Yan, Winston X.; Hunnewell, Pratyusha; Alfonse, Lauren E. Science, Vol. 363, Issue 6422 https://doi.org/10.1126/science.aav7271	journal	December 2018
Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage Stella, Stefano; Alcón, Pablo; Montoya, Guillermo Nature, Vol. 546, Issue 7659 https://doi.org/10.1038/nature22398	journal	May 2017
X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution Putnam, Christopher D.; Hammel, Michal; Hura, Greg L. Quarterly Reviews of Biophysics, Vol. 40, Issue 3 https://doi.org/10.1017/S0033583507004635	journal	August 2007
Accurate SAXS Profile Computation and its Assessment by Contrast Variation Experiments Schneidman-Duhovny, Dina; Hammel, Michal; Tainer, John A. Biophysical Journal, Vol. 105, Issue 4 https://doi.org/10.1016/j.bpj.2013.07.020	journal	August 2013
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
The Discovery, Mechanisms, and Evolutionary Impact of Anti-CRISPRs Borges, Adair L.; Davidson, Alan R.; Bondy-Denomy, Joseph Annual Review of Virology, Vol. 4, Issue 1 https://doi.org/10.1146/annurev-virology-101416-041616	journal	September 2017
An anti-CRISPR protein disables type V Cas12a by acetylation Dong, Liyong; Guan, Xiaoyu; Li, Ningning Nature Structural & Molecular Biology, Vol. 26, Issue 4 https://doi.org/10.1038/s41594-019-0206-1	journal	April 2019
FoXS, FoXSDock and MultiFoXS: Single-state and multi-state structural modeling of proteins and their complexes based on SAXS profiles Schneidman-Duhovny, Dina; Hammel, Michal; Tainer, John A. Nucleic Acids Research, Vol. 44, Issue W1 https://doi.org/10.1093/nar/gkw389	journal	May 2016
Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1 Nishimasu, Hiroshi; Yamano, Takashi; Gao, Linyi Molecular Cell, Vol. 67, Issue 1 https://doi.org/10.1016/j.molcel.2017.04.019	journal	July 2017
An Optimized microRNA Backbone for Effective Single-Copy RNAi Fellmann, Christof; Hoffmann, Thomas; Sridhar, Vaishali Cell Reports, Vol. 5, Issue 6 https://doi.org/10.1016/j.celrep.2013.11.020	journal	December 2013
MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy Zheng, Shawn Q.; Palovcak, Eugene; Armache, Jean-Paul Nature Methods, Vol. 14, Issue 4 https://doi.org/10.1038/nmeth.4193	journal	February 2017
Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems Shmakov, Sergey; Abudayyeh, Omar O.; Makarova, Kira S. Molecular Cell, Vol. 60, Issue 3, p. 385-397 https://doi.org/10.1016/j.molcel.2015.10.008	journal	November 2015
Mechanistic Insights into the cis- and trans-Acting DNase Activities of Cas12a Swarts, Daan C.; Jinek, Martin Molecular Cell, Vol. 73, Issue 3 https://doi.org/10.1016/j.molcel.2018.11.021	journal	February 2019
Real-space refinement in PHENIX for cryo-EM and crystallography Afonine, Pavel V.; Poon, Billy K.; Read, Randy J. Acta Crystallographica Section D Structural Biology, Vol. 74, Issue 6 https://doi.org/10.1107/S2059798318006551	journal	May 2018
SerialEM: A Program for Automated Tilt Series Acquisition on Tecnai Microscopes Using Prediction of Specimen Position Mastronarde, David N. Microscopy and Microanalysis, Vol. 9, Issue S02 https://doi.org/10.1017/S1431927603445911	journal	July 2003
Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS) Hura, Greg L.; Menon, Angeli L.; Hammel, Michal Nature Methods, Vol. 6, Issue 8 https://doi.org/10.1038/nmeth.1353	journal	July 2009
MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability Katoh, K.; Standley, D. M. Molecular Biology and Evolution, Vol. 30, Issue 4 https://doi.org/10.1093/molbev/mst010	journal	January 2013
IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies Nguyen, Lam-Tung; Schmidt, Heiko A.; von Haeseler, Arndt Molecular Biology and Evolution, Vol. 32, Issue 1 https://doi.org/10.1093/molbev/msu300	journal	November 2014
Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a Swarts, Daan C.; van der Oost, John; Jinek, Martin Molecular Cell, Vol. 66, Issue 2 https://doi.org/10.1016/j.molcel.2017.03.016	journal	April 2017
Gctf: Real-time CTF determination and correction Zhang, Kai Journal of Structural Biology, Vol. 193, Issue 1 https://doi.org/10.1016/j.jsb.2015.11.003	journal	January 2016
Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees Letunic, Ivica; Bork, Peer Nucleic Acids Research, Vol. 44, Issue W1 https://doi.org/10.1093/nar/gkw290	journal	April 2016
Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System Zetsche, Bernd; Gootenberg, Jonathan S.; Abudayyeh, Omar O. Cell, Vol. 163, Issue 3, p. 759-771 https://doi.org/10.1016/j.cell.2015.09.038	journal	October 2015
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
Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the Advanced Light Source Classen, Scott; Hura, Greg L.; Holton, James M. Journal of Applied Crystallography, Vol. 46, Issue 1 https://doi.org/10.1107/S0021889812048698	journal	January 2013
Anti-CRISPR: discovery, mechanism and function Pawluk, April; Davidson, Alan R.; Maxwell, Karen L. Nature Reviews Microbiology, Vol. 16, Issue 1 https://doi.org/10.1038/nrmicro.2017.120	journal	October 2017
Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity Stella, Stefano; Mesa, Pablo; Thomsen, Johannes Cell, Vol. 175, Issue 7 https://doi.org/10.1016/j.cell.2018.10.045	journal	December 2018
Enzymatic anti-CRISPRs improve the bacteriophage arsenal Suresh, Shravanti K.; Murugan, Karthik; Sashital, Dipali G. Nature Structural & Molecular Biology, Vol. 26, Issue 4 https://doi.org/10.1038/s41594-019-0210-5	journal	April 2019
High-Throughput SAXS for the Characterization of Biomolecules in Solution: A Practical Approach Dyer, Kevin N.; Hammel, Michal; Rambo, Robert P. Structural Genomics https://doi.org/10.1007/978-1-62703-691-7_18	book	October 2013
MolProbity : all-atom structure validation for macromolecular crystallography Chen, Vincent B.; Arendall, W. Bryan; Headd, Jeffrey J. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 1 https://doi.org/10.1107/S0907444909042073	journal	December 2009
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
The crystal structure of Cpf1 in complex with CRISPR RNA Dong, De; Ren, Kuan; Qiu, Xiaolin Nature, Vol. 532, Issue 7600 https://doi.org/10.1038/nature17944	journal	April 2016
Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection East-Seletsky, Alexandra; O’Connell, Mitchell R.; Knight, Spencer C. Nature, Vol. 538, Issue 7624 https://doi.org/10.1038/nature19802	journal	September 2016
Features and development of Coot Emsley, P.; Lohkamp, B.; Scott, W. G. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/S0907444910007493	journal	March 2010
The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA Fonfara, Ines; Richter, Hagen; Bratovič, Majda Nature, Vol. 532, Issue 7600 https://doi.org/10.1038/nature17945	journal	April 2016
Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1 Yamano, Takashi; Zetsche, Bernd; Ishitani, Ryuichiro Molecular Cell, Vol. 67, Issue 4 https://doi.org/10.1016/j.molcel.2017.06.035	journal	August 2017
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
Structural Variation of Type I-F CRISPR RNA Guided DNA Surveillance Pausch, Patrick; Müller-Esparza, Hanna; Gleditzsch, Daniel Molecular Cell, Vol. 67, Issue 4 https://doi.org/10.1016/j.molcel.2017.06.036	journal	August 2017
PHENIX: a comprehensive Python-based system for macromolecular structure solution Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221 https://doi.org/10.1107/S0907444909052925	journal	January 2010
Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA Yamano, Takashi; Nishimasu, Hiroshi; Zetsche, Bernd Cell, Vol. 165, Issue 4 https://doi.org/10.1016/j.cell.2016.04.003	journal	May 2016
Structure and flexibility within proteins as identified through small angle X-ray scattering Pelikan, M.; Hura, G.; Hammel, M. General Physiology and Biophysics, Vol. 28, Issue 2 https://doi.org/10.4149/gpb_2009_02_174	journal	January 2009
Stock-based detection of protein oligomeric states in jsPISA Krissinel, Eugene Nucleic Acids Research, Vol. 43, Issue W1 https://doi.org/10.1093/nar/gkv314	journal	April 2015
The diversity-generating benefits of a prokaryotic adaptive immune system van Houte, Stineke; Ekroth, Alice K. E.; Broniewski, Jenny M. Nature, Vol. 532, Issue 7599 https://doi.org/10.1038/nature17436	journal	April 2016
cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination Punjani, Ali; Rubinstein, John L.; Fleet, David J. Nature Methods, Vol. 14, Issue 3 https://doi.org/10.1038/nmeth.4169	journal	February 2017
Accurate assessment of mass, models and resolution by small-angle scattering Rambo, Robert P.; Tainer, John A. Nature, Vol. 496, Issue 7446 https://doi.org/10.1038/nature12070	journal	April 2013
CRISPR-Cas guides the future of genetic engineering Knott, Gavin J.; Doudna, Jennifer A. Science, Vol. 361, Issue 6405 https://doi.org/10.1126/science.aat5011	journal	August 2018
Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes Carte, J.; Wang, R.; Li, H. Genes & Development, Vol. 22, Issue 24 https://doi.org/10.1101/gad.1742908	journal	December 2008
Broad-spectrum enzymatic inhibition of CRISPR-Cas12a Knott, Gavin J.; Thornton, Brittney W.; Lobba, Marco J. Nature Structural & Molecular Biology, Vol. 26, Issue 4 https://doi.org/10.1038/s41594-019-0208-z	journal	April 2019
Exploring the gas access routes in a [NiFeSe] hydrogenase using crystals pressurized with krypton and oxygen Zacarias, Sónia; Temporão, Adriana; Carpentier, Philippe JBIC Journal of Biological Inorganic Chemistry, Vol. 25, Issue 6 https://doi.org/10.1007/s00775-020-01814-y	journal	August 2020
Systematic discovery of pseudomonad genetic factors involved in sensitivity to tailocins Carim, Sean; Azadeh, Ashley L.; Kazakov, Alexey E. The ISME Journal, Vol. 15, Issue 8 https://doi.org/10.1038/s41396-021-00921-1	journal	March 2021
PARP1 exhibits enhanced association and catalytic efficiency with γH2A.X-nucleosome Sharma, Deepti; De Falco, Louis; Padavattan, Sivaraman Nature Communications, Vol. 10, Issue 1 https://doi.org/10.1038/s41467-019-13641-0	journal	December 2019
Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation Tillu, Vikas A.; Rae, James; Gao, Ya Nature Communications, Vol. 12, Issue 1 https://doi.org/10.1038/s41467-021-21035-4	journal	February 2021
Insights on autophagosome–lysosome tethering from structural and biochemical characterization of human autophagy factor EPG5 Nam, Sung-Eun; Cheung, Yiu Wing Sunny; Nguyen, Thanh Ngoc Communications Biology, Vol. 4, Issue 1 https://doi.org/10.1038/s42003-021-01830-x	journal	March 2021
PHASTER: a better, faster version of the PHAST phage search tool Arndt, David; Grant, Jason R.; Marcu, Ana Nucleic Acids Research, Vol. 44, Issue W1 https://doi.org/10.1093/nar/gkw387	journal	May 2016
Structural basis for guide RNA processing and seed-dependent DNA targeting by CRISPR-Cas12a Swarts, Daan C.; Van Der Oost, John; Jinek, Martin Cell Press (Elsevier) https://doi.org/10.5167/uzh-138462	text	January 2017
Mechanistic Insights into the cis- and trans-Acting DNase Activities of Cas12a C., Swarts, Daan; Martin, Jinek, Cell Press (Elsevier) https://doi.org/10.5167/uzh-174008	text	January 2019
PHENIX: a comprehensive Python-based system for macromolecular structure solution. Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor Apollo - University of Cambridge Repository https://doi.org/10.17863/cam.45787	text	January 2010
Mechanistic Insights into the Cis- and Trans-acting DNase Activities of Cas12a Swarts, Daan Mendeley https://doi.org/10.17632/dvvxbf5v2p.1	dataset	January 2018

Cited By (3)

Making the cut(s): how Cas12a cleaves target and non-target DNA Swarts, Daan C. Biochemical Society Transactions, Vol. 47, Issue 5 https://doi.org/10.1042/bst20190564	journal	October 2019
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
Machine learning predicts new anti-CRISPR proteins Eitzinger, Simon; Asif, Amina; Watters, Kyle E. Nucleic Acids Research, Vol. 48, Issue 9 https://doi.org/10.1093/nar/gkaa219	journal	April 2020