Genome expansion by a CRISPR trimmer-integrase

Wang, Joy Y.; Tuck, Owen T.; Skopintsev, Petr; Soczek, Katarzyna M.; Li, Gary; Al-Shayeb, Basem; Zhou, Julia; Doudna, Jennifer A.

doi:10.1038/s41586-023-06178-2

Genome expansion by a CRISPR trimmer-integrase

Journal Article · Wed Jun 14 00:00:00 EDT 2023 · Nature (London)

DOI:https://doi.org/10.1038/s41586-023-06178-2· OSTI ID:2470969

Wang, Joy Y. ^[1]; ^[1]; Skopintsev, Petr ^[1]; ^[1]; Li, Gary ^[1]; Al-Shayeb, Basem ^[1]; Zhou, Julia ^[1]; ^[2]

University of California, Berkeley, CA (United States)
University of California, Berkeley, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); University of California, San Francisco, CA (United States)

CRISPR–Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1–Cas2 integrase is necessary but not sufficient. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation, but many CRISPR–Cas systems lack Cas4. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1–Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited exonucleases for faithful acquisition of new CRISPR immune sequences.

View Accepted Manuscript (DOE)

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

Sponsoring Organization:: National Science Foundation (NSF); USDOE

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2470969

Journal Information:: Nature (London), Journal Name: Nature (London) Journal Issue: 7966 Vol. 618; ISSN 0028-0836

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

References (49)

UCSF ChimeraX : Structure visualization for researchers, educators, and developers Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C. Protein Science, Vol. 30, Issue 1 https://doi.org/10.1002/pro.3943	journal	October 2020
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
Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign Genetic Elements Mojica, Francisco J. M.; D�ez-Villase�or, Chc)sar; Garc�a-Mart�nez, Jes�s Journal of Molecular Evolution, Vol. 60, Issue 2 https://doi.org/10.1007/s00239-004-0046-3	journal	February 2005
RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex Hale, Caryn R.; Zhao, Peng; Olson, Sara Cell, Vol. 139, Issue 5 https://doi.org/10.1016/j.cell.2009.07.040	journal	November 2009
Structural and Mechanistic Basis of PAM-Dependent Spacer Acquisition in CRISPR-Cas Systems Wang, Jiuyu; Li, Jiazhi; Zhao, Hongtu Cell, Vol. 163, Issue 4 https://doi.org/10.1016/j.cell.2015.10.008	journal	November 2015
Biology and Applications of CRISPR Systems: Harnessing Nature’s Toolbox for Genome Engineering Wright, Addison V.; Nuñez, James K.; Doudna, Jennifer A. Cell, Vol. 164, Issue 1-2 https://doi.org/10.1016/j.cell.2015.12.035	journal	January 2016
Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation Kieper, Sebastian N.; Almendros, Cristóbal; Behler, Juliane Cell Reports, Vol. 22, Issue 13 https://doi.org/10.1016/j.celrep.2018.02.103	journal	March 2018
3D variability analysis: Resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM Punjani, Ali; Fleet, David J. Journal of Structural Biology, Vol. 213, Issue 2 https://doi.org/10.1016/j.jsb.2021.107702	journal	June 2021
Adaptation in CRISPR-Cas Systems Sternberg, Samuel H.; Richter, Hagen; Charpentier, Emmanuelle Molecular Cell, Vol. 61, Issue 6 https://doi.org/10.1016/j.molcel.2016.01.030	journal	March 2016
CRISPR Immunological Memory Requires a Host Factor for Specificity Nuñez, James K.; Bai, Lawrence; Harrington, Lucas B. Molecular Cell, Vol. 62, Issue 6 https://doi.org/10.1016/j.molcel.2016.04.027	journal	June 2016
Cas4-Dependent Prespacer Processing Ensures High-Fidelity Programming of CRISPR Arrays Lee, Hayun; Zhou, Yi; Taylor, David W. Molecular Cell, Vol. 70, Issue 1 https://doi.org/10.1016/j.molcel.2018.03.003	journal	April 2018
PAM binding ensures orientational integration during Cas4-Cas1-Cas2-mediated CRISPR adaptation Dhingra, Yukti; Suresh, Shravanti K.; Juneja, Puneet Molecular Cell, Vol. 82, Issue 22 https://doi.org/10.1016/j.molcel.2022.09.030	journal	November 2022
CRISPR adaptation from a structural perspective Sasnauskas, Giedrius; Siksnys, Virginijus Current Opinion in Structural Biology, Vol. 65 https://doi.org/10.1016/j.sbi.2020.05.015	journal	December 2020
Self versus non-self discrimination during CRISPR RNA-directed immunity Marraffini, Luciano A.; Sontheimer, Erik J. Nature, Vol. 463, Issue 7280 https://doi.org/10.1038/nature08703	journal	January 2010
The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA Garneau, Josiane E.; Dupuis, Marie-Ève; Villion, Manuela Nature, Vol. 468, Issue 7320 https://doi.org/10.1038/nature09523	journal	November 2010
Integrase-mediated spacer acquisition during CRISPR–Cas adaptive immunity Nuñez, James K.; Lee, Amy S. Y.; Engelman, Alan Nature, Vol. 519, Issue 7542 https://doi.org/10.1038/nature14237	journal	February 2015
Foreign DNA capture during CRISPR–Cas adaptive immunity Nuñez, James K.; Harrington, Lucas B.; Kranzusch, Philip J. Nature, Vol. 527, Issue 7579 https://doi.org/10.1038/nature15760	journal	October 2015
CRISPR–Cas encoding of a digital movie into the genomes of a population of living bacteria Shipman, Seth L.; Nivala, Jeff; Macklis, Jeffrey D. Nature, Vol. 547, Issue 7663 https://doi.org/10.1038/nature23017	journal	July 2017
How type II CRISPR–Cas establish immunity through Cas1–Cas2-mediated spacer integration Xiao, Yibei; Ng, Sherwin; Nam, Ki Hyun Nature, Vol. 550, Issue 7674 https://doi.org/10.1038/nature24020	journal	September 2017
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
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
Cas1–Cas2 complex formation mediates spacer acquisition during CRISPR–Cas adaptive immunity Nuñez, James K.; Kranzusch, Philip J.; Noeske, Jonas Nature Structural & Molecular Biology, Vol. 21, Issue 6 https://doi.org/10.1038/nsmb.2820	journal	May 2014
Protecting genome integrity during CRISPR immune adaptation Wright, Addison V.; Doudna, Jennifer A. Nature Structural & Molecular Biology, Vol. 23, Issue 10 https://doi.org/10.1038/nsmb.3289	journal	September 2016
Molecular mechanisms of CRISPR–Cas spacer acquisition McGinn, Jon; Marraffini, Luciano A. Nature Reviews Microbiology, Vol. 17, Issue 1 https://doi.org/10.1038/s41579-018-0071-7	journal	August 2018
Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants Makarova, Kira S.; Wolf, Yuri I.; Iranzo, Jaime Nature Reviews Microbiology, Vol. 18, Issue 2 https://doi.org/10.1038/s41579-019-0299-x	journal	December 2019
Selective loading and processing of prespacers for precise CRISPR adaptation Kim, Sungchul; Loeff, Luuk; Colombo, Sabina Nature, Vol. 579, Issue 7797 https://doi.org/10.1038/s41586-020-2018-1	journal	February 2020
Highly accurate protein structure prediction with AlphaFold Jumper, John; Evans, Richard; Pritzel, Alexander Nature https://doi.org/10.1038/s41586-021-03819-2	journal	July 2021
Mechanism for Cas4-assisted directional spacer acquisition in CRISPR–Cas Hu, Chunyi; Almendros, Cristóbal; Nam, Ki Hyun Nature, Vol. 598, Issue 7881 https://doi.org/10.1038/s41586-021-03951-z	journal	September 2021
Recording gene expression order in DNA by CRISPR addition of retron barcodes Bhattarai-Kline, Santi; Lear, Sierra K.; Fishman, Chloe B. Nature, Vol. 608, Issue 7921 https://doi.org/10.1038/s41586-022-04994-6	journal	July 2022
Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs Bepler, Tristan; Morin, Andrew; Rapp, Micah Nature Methods, Vol. 16, Issue 11 https://doi.org/10.1038/s41592-019-0575-8	journal	October 2019
Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction Punjani, Ali; Zhang, Haowei; Fleet, David J. Nature Methods, Vol. 17, Issue 12 https://doi.org/10.1038/s41592-020-00990-8	journal	November 2020
Real-time observation of CRISPR spacer acquisition by Cas1–Cas2 integrase Budhathoki, Jagat B.; Xiao, Yibei; Schuler, Gabriel Nature Structural & Molecular Biology, Vol. 27, Issue 5 https://doi.org/10.1038/s41594-020-0415-7	journal	May 2020
Processing and integration of functionally oriented prespacers in the Escherichia coli CRISPR system depends on bacterial host exonucleases Ramachandran, Anita; Summerville, Lesley; Learn, Brian A. Journal of Biological Chemistry, Vol. 295, Issue 11 https://doi.org/10.1074/jbc.RA119.012196	journal	March 2020
PAM identification by CRISPR-Cas effector complexes: diversified mechanisms and structures Gleditzsch, Daniel; Pausch, Patrick; Müller-Esparza, Hanna RNA Biology, Vol. 16, Issue 4 https://doi.org/10.1080/15476286.2018.1504546	journal	September 2018
Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli Yosef, Ido; Goren, Moran G.; Qimron, Udi Nucleic Acids Research, Vol. 40, Issue 12 https://doi.org/10.1093/nar/gks216	journal	February 2012
Short motif sequences determine the targets of the prokaryotic CRISPR defence system Mojica, F. J. M.; Díez-Villaseñor, C.; García-Martínez, J. Microbiology, Vol. 155, Issue 3, p. 733-740 https://doi.org/10.1099/mic.0.023960-0	journal	March 2009
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
Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix Liebschner, Dorothee; Afonine, Pavel V.; Baker, Matthew L. Acta Crystallographica Section D Structural Biology, Vol. 75, Issue 10 https://doi.org/10.1107/S2059798319011471	journal	October 2019
Host nucleases generate prespacers for primed adaptation in the E. coli type I-E CRISPR-Cas system Shiriaeva, Anna A.; Kuznedelov, Konstantin; Fedorov, Ivan Science Advances, Vol. 8, Issue 47 https://doi.org/10.1126/sciadv.abn8650	journal	November 2022
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
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
Molecular recordings by directed CRISPR spacer acquisition Shipman, Seth L.; Nivala, Jeff; Macklis, Jeffrey D. Science, Vol. 353, Issue 6298 https://doi.org/10.1126/science.aaf1175	journal	June 2016
Structures of the CRISPR genome integration complex Wright, Addison V.; Liu, Jun-Jie; Knott, Gavin J. Science, Vol. 357, Issue 6356 https://doi.org/10.1126/science.aao0679	journal	July 2017
Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus Deveau, H.; Barrangou, R.; Garneau, J. E. Journal of Bacteriology, Vol. 190, Issue 4 https://doi.org/10.1128/JB.01412-07	journal	December 2007
HH-suite3 for fast remote homology detection and deep protein annotation Steinegger, Martin; Meier, Markus; Mirdita, Milot BMC Bioinformatics, Vol. 20, Issue 1 https://doi.org/10.1186/s12859-019-3019-7	journal	September 2019
Phylogenomics of Cas4 family nucleases Hudaiberdiev, Sanjarbek; Shmakov, Sergey; Wolf, Yuri I. BMC Evolutionary Biology, Vol. 17, Issue 1 https://doi.org/10.1186/s12862-017-1081-1	journal	November 2017
DnaQ exonuclease‐like domain of Cas2 promotes spacer integration in a type I‐E CRISPR‐Cas system Drabavicius, Gediminas; Sinkunas, Tomas; Silanskas, Arunas EMBO reports, Vol. 19, Issue 7 https://doi.org/10.15252/embr.201745543	journal	May 2018
CRISPRDetect: A flexible algorithm to define CRISPR arrays Biswas, Ambarish; Staals, Raymond; Morales, Sergio Figshare https://doi.org/10.6084/m9.figshare.c.3618623.v1	collection	January 2016
The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation Lee, Hayun; Dhingra, Yukti; Sashital, Dipali G. eLife, Vol. 8 https://doi.org/10.7554/eLife.44248	journal	April 2019

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