The CRISPR RNA-guided surveillance complex in Escherichia coli accommodates extended RNA spacers

Luo, Michelle L.; Jackson, Ryan N.; Denny, Steven R.; Tokmina-Lukaszewska, Monika; Maksimchuk, Kenneth R.; Lin, Wayne; Bothner, Brian; Wiedenheft, Blake; Beisel, Chase L.

doi:10.1093/nar/gkw421

Title: The CRISPR RNA-guided surveillance complex in Escherichia coli accommodates extended RNA spacers

Journal Article · Thu May 12 00:00:00 EDT 2016 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gkw421· OSTI ID:1360145

Luo, Michelle L. ^[1]; Jackson, Ryan N. ^[2]; Denny, Steven R. ^[1]; Tokmina-Lukaszewska, Monika ^[2]; Maksimchuk, Kenneth R. ^[1]; Lin, Wayne ^[2]; Bothner, Brian ^[2]; Wiedenheft, Blake ^[2]; Beisel, Chase L. ^[1]

North Carolina State Univ., Raleigh, NC (United States)
Montana State Univ., Bozeman, MT (United States)

Bacteria and archaea acquire resistance to foreign genetic elements by integrating fragments of foreign DNA into CRISPR (clustered regularly interspaced short palindromic repeats) loci. In Escherichia coli, CRISPR-derived RNAs (crRNAs) assemble with Cas proteins into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade recognizes DNA targets via protein-mediated recognition of a protospacer adjacent motif and complementary base pairing between the crRNA spacer and the DNA target. Previously determined structures of Cascade showed that the crRNA is stretched along an oligomeric protein assembly, leading us to ask how crRNA length impacts the assembly and function of this complex. We found that extending the spacer portion of the crRNA resulted in larger Cascade complexes with altered stoichiometry and preserved in vitro binding affinity for target DNA. Longer spacers also preserved the in vivo ability of Cascade to repress target gene expression and to recruit the Cas3 endonuclease for target degradation. Lastly, longer spacers exhibited enhanced silencing at particular target locations and were sensitive to mismatches within the extended region. These findings demonstrate the flexibility of the Type I-E CRISPR machinery and suggest that spacer length can be modified to fine-tune Cascade activity.

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Research Organization:: Univ. of Colorado, Boulder, CO (United States)

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: SC0012518

OSTI ID:: 1360145

Journal Information:: Nucleic Acids Research, Vol. 44, Issue 15; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 34 works

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References (62)

Unravelling the structural and mechanistic basis of CRISPR–Cas systems van der Oost, John; Westra, Edze R.; Jackson, Ryan N. Nature Reviews Microbiology, Vol. 12, Issue 7 https://doi.org/10.1038/nrmicro3279	journal	June 2014
An updated evolutionary classification of CRISPR–Cas systems Makarova, Kira S.; Wolf, Yuri I.; Alkhnbashi, Omer S. Nature Reviews Microbiology, Vol. 13, Issue 11, p. 722-736 https://doi.org/10.1038/nrmicro3569	journal	September 2015
CRISPR-based adaptive and heritable immunity in prokaryotes van der Oost, John; Jore, Matthijs M.; Westra, Edze R. Trends in Biochemical Sciences, Vol. 34, Issue 8 https://doi.org/10.1016/j.tibs.2009.05.002	journal	August 2009
CRISPR-Cas Systems in Bacteria and Archaea: Versatile Small RNAs for Adaptive Defense and Regulation Bhaya, Devaki; Davison, Michelle; Barrangou, Rodolphe Annual Review of Genetics, Vol. 45, Issue 1, p. 273-297 https://doi.org/10.1146/annurev-genet-110410-132430	journal	December 2011
RNA-guided genetic silencing systems in bacteria and archaea Wiedenheft, Blake; Sternberg, Samuel H.; Doudna, Jennifer A. Nature, Vol. 482, Issue 7385, p. 331-338 https://doi.org/10.1038/nature10886	journal	February 2012
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
Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system: Cas3 nuclease/helicase Sinkunas, Tomas; Gasiunas, Giedrius; Fremaux, Christophe The EMBO Journal, Vol. 30, Issue 7 https://doi.org/10.1038/emboj.2011.41	journal	February 2011
Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation Huo, Yanwu; Nam, Ki Hyun; Ding, Fang Nature Structural & Molecular Biology, Vol. 21, Issue 9 https://doi.org/10.1038/nsmb.2875	journal	August 2014
CRISPR Immunity Relies on the Consecutive Binding and Degradation of Negatively Supercoiled Invader DNA by Cascade and Cas3 Westra, Edze R.; van Erp, Paul B. G.; Künne, Tim Molecular Cell, Vol. 46, Issue 5 https://doi.org/10.1016/j.molcel.2012.03.018	journal	June 2012
Fitting CRISPR-associated Cas3 into the Helicase Family Tree Jackson, Ryan N.; Lavin, Matthew; Carter, Joshua Current Opinion in Structural Biology, Vol. 24 https://doi.org/10.1016/j.sbi.2014.01.001	journal	February 2014
In Vitro Reconstitution of an Escherichia coli RNA-guided Immune System Reveals Unidirectional, ATP-dependent Degradation of DNA Target Mulepati, Sabin; Bailey, Scott Journal of Biological Chemistry, Vol. 288, Issue 31 https://doi.org/10.1074/jbc.M113.472233	journal	June 2013
Structural basis for CRISPR RNA-guided DNA recognition by Cascade Jore, Matthijs M.; Lundgren, Magnus; van Duijn, Esther Nature Structural & Molecular Biology, Vol. 18, Issue 5 https://doi.org/10.1038/nsmb.2019	journal	April 2011
Surveillance and Processing of Foreign DNA by the Escherichia coli CRISPR-Cas System Redding, Sy; Sternberg, Samuel H.; Marshall, Myles Cell, Vol. 163, Issue 4 https://doi.org/10.1016/j.cell.2015.10.003	journal	November 2015
Structural basis for promiscuous PAM recognition in type I–E Cascade from E. coli Hayes, Robert P.; Xiao, Yibei; Ding, Fran Nature, Vol. 530, Issue 7591 https://doi.org/10.1038/nature16995	journal	February 2016
Crystal structure of the CRISPR RNA-guided surveillance complex from Escherichia coli Jackson, R. N.; Golden, S. M.; van Erp, P. B. G. Science, Vol. 345, Issue 6203 https://doi.org/10.1126/science.1256328	journal	August 2014
Crystal structure of the RNA-guided immune surveillance Cascade complex in Escherichia coli Zhao, Hongtu; Sheng, Gang; Wang, Jiuyu Nature, Vol. 515, Issue 7525 https://doi.org/10.1038/nature13733	journal	August 2014
Structures of the RNA-guided surveillance complex from a bacterial immune system Wiedenheft, Blake; Lander, Gabriel C.; Zhou, Kaihong Nature, Vol. 477, Issue 7365 https://doi.org/10.1038/nature10402	journal	September 2011
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
In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus Sinkunas, Tomas; Gasiunas, Giedrius; Waghmare, Sakharam P. The EMBO Journal, Vol. 32, Issue 3 https://doi.org/10.1038/emboj.2012.352	journal	January 2013
Current and future prospects for CRISPR-based tools in bacteria: CRISPR-Based Tools in Bacteria Luo, Michelle L.; Leenay, Ryan T.; Beisel, Chase L. Biotechnology and Bioengineering, Vol. 113, Issue 5 https://doi.org/10.1002/bit.25851	journal	October 2015
Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression Luo, Michelle L.; Mullis, Adam S.; Leenay, Ryan T. Nucleic Acids Research, Vol. 43, Issue 1 https://doi.org/10.1093/nar/gku971	journal	October 2014
Efficient programmable gene silencing by Cascade Rath, Devashish; Amlinger, Lina; Hoekzema, Mirthe Nucleic Acids Research, Vol. 43, Issue 1 https://doi.org/10.1093/nar/gku1257	journal	November 2014
Programmable Removal of Bacterial Strains by Use of Genome-Targeting CRISPR-Cas Systems Gomaa, Ahmed A.; Klumpe, Heidi E.; Luo, Michelle L. mBio, Vol. 5, Issue 1 https://doi.org/10.1128/mBio.00928-13	journal	January 2014
Targeted DNA degradation using a CRISPR device stably carried in the host genome Caliando, Brian J.; Voigt, Christopher A. Nature Communications, Vol. 6, Issue 1 https://doi.org/10.1038/ncomms7989	journal	May 2015
Harnessing Type I and Type III CRISPR-Cas systems for genome editing Li, Yingjun; Pan, Saifu; Zhang, Yan Nucleic Acids Research, Vol. 44, Issue 4 https://doi.org/10.1093/nar/gkv1044	journal	October 2015
Diversity of CRISPR loci in Escherichia coli Díez-Villaseñor, C.; Almendros, C.; García-Martínez, J. Microbiology, Vol. 156, Issue 5 https://doi.org/10.1099/mic.0.036046-0	journal	May 2010
Crystal structure of a CRISPR RNA-guided surveillance complex bound to a ssDNA target Mulepati, S.; Heroux, A.; Bailey, S. Science, Vol. 345, Issue 6203 https://doi.org/10.1126/science.1256996	journal	August 2014
Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant Cherepanov, Peter P.; Wackernagel, Wilfried Gene, Vol. 158, Issue 1 https://doi.org/10.1016/0378-1119(95)00193-A	journal	January 1995
A comprehensive library of fluorescent transcriptional reporters for Escherichia coli Zaslaver, Alon; Bren, Anat; Ronen, Michal Nature Methods, Vol. 3, Issue 8, p. 623-628 https://doi.org/10.1038/nmeth895	journal	August 2006
Blue native PAGE Wittig, Ilka; Braun, Hans-Peter; Schägger, Hermann Nature Protocols, Vol. 1, Issue 1 https://doi.org/10.1038/nprot.2006.62	journal	June 2006
Type I-E CRISPR-Cas Systems Discriminate Target from Non-Target DNA through Base Pairing-Independent PAM Recognition Westra, Edze R.; Semenova, Ekaterina; Datsenko, Kirill A. PLoS Genetics, Vol. 9, Issue 9 https://doi.org/10.1371/journal.pgen.1003742	journal	September 2013
Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems Leenay, Ryan T.; Maksimchuk, Kenneth R.; Slotkowski, Rebecca A. Molecular Cell, Vol. 62, Issue 1 https://doi.org/10.1016/j.molcel.2016.02.031	journal	April 2016
Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli van Erp, Paul B. G.; Jackson, Ryan N.; Carter, Joshua Nucleic Acids Research, Vol. 43, Issue 17 https://doi.org/10.1093/nar/gkv793	journal	August 2015
Directional R-Loop Formation by the CRISPR-Cas Surveillance Complex Cascade Provides Efficient Off-Target Site Rejection Rutkauskas, Marius; Sinkunas, Tomas; Songailiene, Inga Cell Reports, Vol. 10, Issue 9 https://doi.org/10.1016/j.celrep.2015.01.067	journal	March 2015
Cytotoxic Chromosomal Targeting by CRISPR/Cas Systems Can Reshape Bacterial Genomes and Expel or Remodel Pathogenicity Islands Vercoe, Reuben B.; Chang, James T.; Dy, Ron L. PLoS Genetics, Vol. 9, Issue 4 https://doi.org/10.1371/journal.pgen.1003454	journal	April 2013
Next Generation Prokaryotic Engineering: The CRISPR-Cas Toolkit Mougiakos, Ioannis; Bosma, Elleke F.; de Vos, Willem M. Trends in Biotechnology, Vol. 34, Issue 7 https://doi.org/10.1016/j.tibtech.2016.02.004	journal	July 2016
Mechanism of Foreign DNA Selection in a Bacterial Adaptive Immune System Sashital, Dipali G.; Wiedenheft, Blake; Doudna, Jennifer A. Molecular Cell, Vol. 46, Issue 5 https://doi.org/10.1016/j.molcel.2012.03.020	journal	June 2012
Two Distinct DNA Binding Modes Guide Dual Roles of a CRISPR-Cas Protein Complex Blosser, Timothy R.; Loeff, Luuk; Westra, Edze R. Molecular Cell, Vol. 58, Issue 1 https://doi.org/10.1016/j.molcel.2015.01.028	journal	April 2015
Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes Szczelkun, Mark D.; Tikhomirova, Maria S.; Sinkunas, Tomas Proceedings of the National Academy of Sciences, Vol. 111, Issue 27 https://doi.org/10.1073/pnas.1402597111	journal	May 2014
Degenerate target sites mediate rapid primed CRISPR adaptation Fineran, P. C.; Gerritzen, M. J. H.; Suarez-Diez, M. Proceedings of the National Academy of Sciences, Vol. 111, Issue 16 https://doi.org/10.1073/pnas.1400071111	journal	April 2014
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
Detection and characterization of spacer integration intermediates in type I-E CRISPR–Cas system Arslan, Zihni; Hermanns, Veronica; Wurm, Reinhild Nucleic Acids Research, Vol. 42, Issue 12 https://doi.org/10.1093/nar/gku510	journal	June 2014
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
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
A Conserved Structural Chassis for Mounting Versatile CRISPR RNA-Guided Immune Responses Jackson, Ryan N.; Wiedenheft, Blake Molecular Cell, Vol. 58, Issue 5 https://doi.org/10.1016/j.molcel.2015.05.023	journal	June 2015
Programmable RNA Shredding by the Type III-A CRISPR-Cas System of Streptococcus thermophilus Tamulaitis, Gintautas; Kazlauskiene, Migle; Manakova, Elena Molecular Cell, Vol. 56, Issue 4 https://doi.org/10.1016/j.molcel.2014.09.027	journal	November 2014
Structure and Activity of the RNA-Targeting Type III-B CRISPR-Cas Complex of Thermus thermophilus Staals, Raymond H. J.; Agari, Yoshihiro; Maki-Yonekura, Saori Molecular Cell, Vol. 52, Issue 1 https://doi.org/10.1016/j.molcel.2013.09.013	journal	October 2013
Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus Hale, C.; Kleppe, K.; Terns, R. M. RNA, Vol. 14, Issue 12 https://doi.org/10.1261/rna.1246808	journal	October 2008
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
Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity Ran, F. Ann; Hsu, Patrick D.; Lin, Chie-Yu Cell, Vol. 154, Issue 6, p. 1380-1389 https://doi.org/10.1016/j.cell.2013.08.021	journal	September 2013
Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display Shechner, David M.; Hacisuleyman, Ezgi; Younger, Scott T. Nature Methods, Vol. 12, Issue 7 https://doi.org/10.1038/nmeth.3433	journal	June 2015
Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression Qi, Lei S.; Larson, Matthew H.; Gilbert, Luke A. Cell, Vol. 152, Issue 5, p. 1173-1183 https://doi.org/10.1016/j.cell.2013.02.022	journal	February 2013
CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering Mali, Prashant; Aach, John; Stranges, P. Benjamin Nature Biotechnology, Vol. 31, Issue 9 https://doi.org/10.1038/nbt.2675	journal	August 2013
Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease Dahlman, James E.; Abudayyeh, Omar O.; Joung, Julia Nature Biotechnology, Vol. 33, Issue 11 https://doi.org/10.1038/nbt.3390	journal	October 2015
Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds Zalatan, Jesse G.; Lee, Michael E.; Almeida, Ricardo Cell, Vol. 160, Issue 1-2 https://doi.org/10.1016/j.cell.2014.11.052	journal	January 2015
RNAi: Prokaryotes Get in on the Act van der Oost, John; Brouns, Stan J. J. Cell, Vol. 139, Issue 5 https://doi.org/10.1016/j.cell.2009.11.018	journal	November 2009
Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity Ran, F. Ann; Hsu, Patrick D.; Lin, Chie-Yu Cell, Vol. 155, Issue 2 https://doi.org/10.1016/j.cell.2013.09.040	journal	October 2013
CRISPR silencing de Souza, Natalie Nature Methods, Vol. 10, Issue 5 https://doi.org/10.1038/nmeth.2466	journal	April 2013
Complex regulatory control with CRISPR de Souza, Natalie Nature Methods, Vol. 12, Issue 3 https://doi.org/10.1038/nmeth.3308	journal	February 2015
Fulfilling the dream of a perfect genome editing tool Spies, Maria Proceedings of the National Academy of Sciences, Vol. 111, Issue 28 https://doi.org/10.1073/pnas.1408985111	journal	July 2014
Analyzing Large Protein Complexes by Structural Mass Spectrometry Kirshenbaum, Noam; Michaelevski, Izhak; Sharon, Michal Journal of Visualized Experiments, Issue 40 https://doi.org/10.3791/1954	journal	January 2010
T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis Michaelevski, Izhak; Kirshenbaum, Noam; Sharon, Michal Journal of Visualized Experiments, Issue 41 https://doi.org/10.3791/1985-v	journal	July 2010

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