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Martínez-Alvarez L, Stickel D, Salegi-Díez A, Bhoobalan-Chitty Y, Shen WZ, Peng X. Natively expressed AcrIII-1 does not function as an anti-CRISPR protein. Nature 2025; 640:E8-E14. [PMID: 40240860 DOI: 10.1038/s41586-025-08649-0] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2023] [Accepted: 01/14/2025] [Indexed: 04/18/2025]
Affiliation(s)
| | - Dominic Stickel
- Department of Biology, University of Copenhagen, Copenhagen, Denmark
| | | | | | | | - Xu Peng
- Department of Biology, University of Copenhagen, Copenhagen, Denmark.
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2
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Karneyeva K, Kolesnik M, Livenskyi A, Zgoda V, Zubarev V, Trofimova A, Artamonova D, Ispolatov Y, Severinov K. Interference Requirements of Type III CRISPR-Cas Systems from Thermus thermophilus. J Mol Biol 2024; 436:168448. [PMID: 38266982 DOI: 10.1016/j.jmb.2024.168448] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2023] [Revised: 01/12/2024] [Accepted: 01/13/2024] [Indexed: 01/26/2024]
Abstract
Among the diverse prokaryotic adaptive immunity mechanisms, the Type III CRISPR-Cas systems are the most complex. The multisubunit Type III effectors recognize RNA targets complementary to CRISPR RNAs (crRNAs). Target recognition causes synthesis of cyclic oligoadenylates that activate downstream auxiliary effectors, which affect cell physiology in complex and poorly understood ways. Here, we studied the ability of III-A and III-B CRISPR-Cas subtypes from Thermus thermophilus to interfere with plasmid transformation. We find that for both systems, requirements for crRNA-target complementarity sufficient for interference depend on the target transcript abundance, with more abundant targets requiring shorter complementarity segments. This result and thermodynamic calculations indicate that Type III effectors bind their targets in a simple bimolecular reaction with more extensive crRNA-target base pairing compensating for lower target abundance. Since the targeted RNA used in our work is non-essential for either the host or the plasmid, the results also establish that a certain number of target-bound effector complexes must be present in the cell to interfere with plasmid establishment. For the more active III-A system, we determine the minimal length of RNA-duplex sufficient for interference and show that the position of this minimal duplex can vary within the effector. Finally, we show that the III-A immunity is dependent on the HD nuclease domain of the Cas10 subunit. Since this domain is absent from the III-B system the result implies that the T. thermophilus III-B system must elicit a more efficient cyclic oligoadenylate-dependent response to provide the immunity.
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Affiliation(s)
- Karyna Karneyeva
- Center for Molecular and Cellular Biology, Skolkovo Institute of Science and Technology, Moscow 121205, Russia
| | - Matvey Kolesnik
- Center for Molecular and Cellular Biology, Skolkovo Institute of Science and Technology, Moscow 121205, Russia
| | - Alexei Livenskyi
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119992, Russia
| | - Viktor Zgoda
- Institute of Biomedical Chemistry, Moscow 119435, Russia
| | - Vasiliy Zubarev
- Center for Molecular and Cellular Biology, Skolkovo Institute of Science and Technology, Moscow 121205, Russia
| | - Anna Trofimova
- Laboratory of Molecular Genetics of Microorganisms, Institute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia
| | - Daria Artamonova
- Center for Molecular and Cellular Biology, Skolkovo Institute of Science and Technology, Moscow 121205, Russia
| | - Yaroslav Ispolatov
- Departamento de Física, Center for Interdisciplinary Research in Astrophysics and Space Science, Universidad de Santiago de Chile, Victor Jara 3493, Santiago, Chile
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3
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Du L, Zhu Q, Lin Z. Molecular mechanism of allosteric activation of the CRISPR ribonuclease Csm6 by cyclic tetra-adenylate. EMBO J 2024; 43:304-315. [PMID: 38177499 PMCID: PMC10897365 DOI: 10.1038/s44318-023-00017-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2023] [Revised: 11/16/2023] [Accepted: 11/27/2023] [Indexed: 01/06/2024] Open
Abstract
Type III CRISPR systems are innate immune systems found in bacteria and archaea, which produce cyclic oligoadenylate (cOA) second messengers in response to viral infections. In these systems, Csm6 proteins serve as ancillary nucleases that degrade single-stranded RNA (ssRNA) upon activation by cOA. In addition, Csm6 proteins also possess cOA-degrading activity as an intrinsic off-switch to avoid degradation of host RNA and DNA that would eventually lead to cell dormancy or cell death. Here, we present the crystal structures of Thermus thermophilus (Tt) Csm6 alone, and in complex with cyclic tetra-adenylate (cA4) in both pre- and post-cleavage states. These structures establish the molecular basis of the long-range allosteric activation of TtCsm6 ribonuclease by cA4. cA4 binding induces significant conformational changes, including closure of the CARF domain, dimerization of the HTH domain, and reorganization of the R-X4-6-H motif within the HEPN domain. The cleavage of cA4 by the CARF domain restores each domain to a conformation similar to its apo state. Furthermore, we have identified hyperactive TtCsm6 variants that exhibit sustained cA4-activated RNase activity, showing great promise for their applications in genome editing and diagnostics.
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Affiliation(s)
- Liyang Du
- College of Chemistry, Fuzhou University, 350108, Fuzhou, China
| | - Qinwei Zhu
- College of Chemistry, Fuzhou University, 350108, Fuzhou, China
| | - Zhonghui Lin
- College of Chemistry, Fuzhou University, 350108, Fuzhou, China.
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4
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Yu Z, Xu J, Zhang Y, She Q. The influence of the copy number of invader on the fate of bacterial host cells in the antiviral defense by CRISPR-Cas10 DNases. ENGINEERING MICROBIOLOGY 2023; 3:100102. [PMID: 39628911 PMCID: PMC11610955 DOI: 10.1016/j.engmic.2023.100102] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 04/03/2023] [Revised: 05/14/2023] [Accepted: 06/19/2023] [Indexed: 12/06/2024]
Abstract
Type III CRISPR-Cas10 systems employ multiple immune activities to defend their hosts against invasion from mobile genetic elements (MGEs), including DNase and cyclic oligoadenylates (cOA) synthesis both of which are hosted by the type-specific protein Cas10. Extensive investigations conducted for the activation of Cas accessory proteins by cOAs have revealed their functions in the type III immunity, but the function of the Cas10 DNase in the same process remains elusive. Here, Lactobacillus delbrueckii subsp. Bulgaricus type III-A (Ld) Csm system, a type III CRISPR system that solely relies on its Cas10 DNase for providing immunity, was employed as a model to investigate the DNase function. Interference assay was conducted in Escherichia coli using two plasmids: pCas carrying the LdCsm system and pTarget producing target RNAs. The former functioned as a de facto "CRISPR host element" while the latter, mimicking an invading MGE. We found that, upon induction of immune responses, the fate of each genetic element was determined by their copy numbers: plasmid of a low copy number was selectively eliminated from the E. coli cells regardless whether it represents a de facto CRISPR host or an invader. Together, we reveal, for the first time, that the immune mechanisms of Cas10 DNases are of two folds: the DNase activity is capable of removing low-copy invaders from infected cells, but it also leads to abortive infection when the invader copy number is high.
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Affiliation(s)
- Zhenxiao Yu
- CRISPR and Archaea Biology Research Center, State Key Laboratory of Microbial Technology and Microbial Technology Institute, Shandong University, Qingdao 266237, China
| | - Jianan Xu
- CRISPR and Archaea Biology Research Center, State Key Laboratory of Microbial Technology and Microbial Technology Institute, Shandong University, Qingdao 266237, China
| | - Yan Zhang
- College of Food and Biological Engineering, Henan University of Animal Husbandry and Economy, Zhengzhou 450000, China
| | - Qunxin She
- CRISPR and Archaea Biology Research Center, State Key Laboratory of Microbial Technology and Microbial Technology Institute, Shandong University, Qingdao 266237, China
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5
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Booker AE, D'Angelo T, Adams-Beyea A, Brown JM, Nigro O, Rappé MS, Stepanauskas R, Orcutt BN. Life strategies for Aminicenantia in subseafloor oceanic crust. THE ISME JOURNAL 2023; 17:1406-1415. [PMID: 37328571 PMCID: PMC10432499 DOI: 10.1038/s41396-023-01454-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/19/2022] [Revised: 04/11/2023] [Accepted: 04/17/2023] [Indexed: 06/18/2023]
Abstract
After decades studying the microbial "deep biosphere" in subseafloor oceanic crust, the growth and life strategies in this anoxic, low energy habitat remain poorly described. Using both single cell genomics and metagenomics, we reveal the life strategies of two distinct lineages of uncultivated Aminicenantia bacteria from the basaltic subseafloor oceanic crust of the eastern flank of the Juan de Fuca Ridge. Both lineages appear adapted to scavenge organic carbon, as each have genetic potential to catabolize amino acids and fatty acids, aligning with previous Aminicenantia reports. Given the organic carbon limitation in this habitat, seawater recharge and necromass may be important carbon sources for heterotrophic microorganisms inhabiting the ocean crust. Both lineages generate ATP via several mechanisms including substrate-level phosphorylation, anaerobic respiration, and electron bifurcation driving an Rnf ion translocation membrane complex. Genomic comparisons suggest these Aminicenantia transfer electrons extracellularly, perhaps to iron or sulfur oxides consistent with mineralogy of this site. One lineage, called JdFR-78, has small genomes that are basal to the Aminicenantia class and potentially use "primordial" siroheme biosynthetic intermediates for heme synthesis, suggesting this lineage retain characteristics of early evolved life. Lineage JdFR-78 contains CRISPR-Cas defenses to evade viruses, while other lineages contain prophage that may help prevent super-infection or no detectable viral defenses. Overall, genomic evidence points to Aminicenantia being well adapted to oceanic crust environments by taking advantage of simple organic molecules and extracellular electron transport.
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Affiliation(s)
- Anne E Booker
- Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, USA
| | | | - Annabelle Adams-Beyea
- Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, USA
- Eugene Lang College of Liberal Arts at The New School, New York City, NY, USA
| | - Julia M Brown
- Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, USA
| | - Olivia Nigro
- Department of Natural Science, Hawai'i Pacific University, Honolulu, HI, USA
| | - Michael S Rappé
- Hawai'i Institute of Marine Biology, SOEST, University of Hawai'i at Mānoa, Kāne'ohe, HI, USA
| | | | - Beth N Orcutt
- Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, USA.
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6
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Zhang Y, Lin J, Tian X, Wang Y, Zhao R, Wu C, Wang X, Zhao P, Bi X, Yu Z, Han W, Peng N, Liang YX, She Q. Inactivation of Target RNA Cleavage of a III-B CRISPR-Cas System Induces Robust Autoimmunity in Saccharolobus islandicus. Int J Mol Sci 2022; 23:ijms23158515. [PMID: 35955649 PMCID: PMC9368842 DOI: 10.3390/ijms23158515] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2022] [Revised: 07/23/2022] [Accepted: 07/28/2022] [Indexed: 12/04/2022] Open
Abstract
Type III CRISPR-Cas systems show the target (tg)RNA-activated indiscriminate DNA cleavage and synthesis of oligoadenylates (cOA) and a secondary signal that activates downstream nuclease effectors to exert indiscriminate RNA/DNA cleavage, and both activities are regulated in a spatiotemporal fashion. In III-B Cmr systems, cognate tgRNAs activate the two Cmr2-based activities, which are then inactivated via tgRNA cleavage by Cmr4, but how Cmr4 nuclease regulates the Cmr immunization remains to be experimentally characterized. Here, we conducted mutagenesis of Cmr4 conserved amino acids in Saccharolobus islandicus, and this revealed that Cmr4α RNase-dead (dCmr4α) mutation yields cell dormancy/death. We also found that plasmid-borne expression of dCmr4α in the wild-type strain strongly reduced plasmid transformation efficiency, and deletion of CRISPR arrays in the host genome reversed the dCmr4α inhibition. Expression of dCmr4α also strongly inhibited plasmid transformation with Cmr2αHD and Cmr2αPalm mutants, but the inhibition was diminished in Cmr2αHD,Palm. Since dCmr4α-containing effectors lack spatiotemporal regulation, this allows an everlasting interaction between crRNA and cellular RNAs to occur. As a result, some cellular RNAs, which are not effective in mediating immunity due to the presence of spatiotemporal regulation, trigger autoimmunity of the Cmr-α system in the S. islandicus cells expressing dCmr4α. Together, these results pinpoint the crucial importance of tgRNA cleavage in autoimmunity avoidance and in the regulation of immunization of type III systems.
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Affiliation(s)
- Yan Zhang
- Henan Engineering Laboratory for Bioconversion Technology of Functional Microbes, College of Life Sciences, Henan Normal University, Xinxiang 453007, China;
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; (X.T.); (Y.W.); (R.Z.); (W.H.); (N.P.); (Y.X.L.)
| | - Jinzhong Lin
- Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark;
| | - Xuhui Tian
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; (X.T.); (Y.W.); (R.Z.); (W.H.); (N.P.); (Y.X.L.)
| | - Yuan Wang
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; (X.T.); (Y.W.); (R.Z.); (W.H.); (N.P.); (Y.X.L.)
| | - Ruiliang Zhao
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; (X.T.); (Y.W.); (R.Z.); (W.H.); (N.P.); (Y.X.L.)
| | - Chenwei Wu
- CRISPR and Archaea Biology Research Center, State Key Laboratory of Microbial Technology, Shandong University, 72 Binhai Road, Qingdao 266237, China; (C.W.); (X.W.); (P.Z.); (X.B.); (Z.Y.)
| | - Xiaoning Wang
- CRISPR and Archaea Biology Research Center, State Key Laboratory of Microbial Technology, Shandong University, 72 Binhai Road, Qingdao 266237, China; (C.W.); (X.W.); (P.Z.); (X.B.); (Z.Y.)
| | - Pengpeng Zhao
- CRISPR and Archaea Biology Research Center, State Key Laboratory of Microbial Technology, Shandong University, 72 Binhai Road, Qingdao 266237, China; (C.W.); (X.W.); (P.Z.); (X.B.); (Z.Y.)
| | - Xiaonan Bi
- CRISPR and Archaea Biology Research Center, State Key Laboratory of Microbial Technology, Shandong University, 72 Binhai Road, Qingdao 266237, China; (C.W.); (X.W.); (P.Z.); (X.B.); (Z.Y.)
| | - Zhenxiao Yu
- CRISPR and Archaea Biology Research Center, State Key Laboratory of Microbial Technology, Shandong University, 72 Binhai Road, Qingdao 266237, China; (C.W.); (X.W.); (P.Z.); (X.B.); (Z.Y.)
| | - Wenyuan Han
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; (X.T.); (Y.W.); (R.Z.); (W.H.); (N.P.); (Y.X.L.)
| | - Nan Peng
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; (X.T.); (Y.W.); (R.Z.); (W.H.); (N.P.); (Y.X.L.)
| | - Yun Xiang Liang
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; (X.T.); (Y.W.); (R.Z.); (W.H.); (N.P.); (Y.X.L.)
| | - Qunxin She
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; (X.T.); (Y.W.); (R.Z.); (W.H.); (N.P.); (Y.X.L.)
- Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark;
- CRISPR and Archaea Biology Research Center, State Key Laboratory of Microbial Technology, Shandong University, 72 Binhai Road, Qingdao 266237, China; (C.W.); (X.W.); (P.Z.); (X.B.); (Z.Y.)
- Correspondence:
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7
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Huang YY, Zhang XY, Zhu P, Ji L. Development of clustered regularly interspaced short palindromic repeats/CRISPR-associated technology for potential clinical applications. World J Clin Cases 2022; 10:5934-5945. [PMID: 35949837 PMCID: PMC9254185 DOI: 10.12998/wjcc.v10.i18.5934] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/03/2021] [Revised: 01/10/2022] [Accepted: 04/24/2022] [Indexed: 02/06/2023] Open
Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) proteins constitute the innate adaptive immune system in several bacteria and archaea. This immune system helps them in resisting the invasion of phages and foreign DNA by providing sequence-specific acquired immunity. Owing to the numerous advantages such as ease of use, low cost, high efficiency, good accuracy, and a diverse range of applications, the CRISPR-Cas system has become the most widely used genome editing technology. Hence, the advent of the CRISPR/Cas technology highlights a tremendous potential in clinical diagnosis and could become a powerful asset for modern medicine. This study reviews the recently reported application platforms for screening, diagnosis, and treatment of different diseases based on CRISPR/Cas systems. The limitations, current challenges, and future prospectus are summarized; this article would be a valuable reference for future genome-editing practices.
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Affiliation(s)
- Yue-Ying Huang
- School of Medical Laboratory, Weifang Medical University, Weifang 261053, Shandong Province, China
| | - Xiao-Yu Zhang
- School of Medical Laboratory, Weifang Medical University, Weifang 261053, Shandong Province, China
| | - Ping Zhu
- School of Medical Laboratory, Weifang Medical University, Weifang 261053, Shandong Province, China
| | - Ling Ji
- Department of Laboratory Medicine, Peking University Shenzhen Hospital, Shenzhen 518035, Guangdong Province, China
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8
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Chou-Zheng L, Hatoum-Aslan A. Critical roles for 'housekeeping' nucleases in type III CRISPR-Cas immunity. eLife 2022; 11:81897. [PMID: 36479971 PMCID: PMC9762709 DOI: 10.7554/elife.81897] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2022] [Accepted: 12/07/2022] [Indexed: 12/13/2022] Open
Abstract
CRISPR-Cas systems are a family of adaptive immune systems that use small CRISPR RNAs (crRNAs) and CRISPR-associated (Cas) nucleases to protect prokaryotes from invading plasmids and viruses (i.e., phages). Type III systems launch a multilayered immune response that relies upon both Cas and non-Cas cellular nucleases, and although the functions of Cas components have been well described, the identities and roles of non-Cas participants remain poorly understood. Previously, we showed that the type III-A CRISPR-Cas system in Staphylococcus epidermidis employs two degradosome-associated nucleases, PNPase and RNase J2, to promote crRNA maturation and eliminate invading nucleic acids (Chou-Zheng and Hatoum-Aslan, 2019). Here, we identify RNase R as a third 'housekeeping' nuclease critical for immunity. We show that RNase R works in concert with PNPase to complete crRNA maturation and identify specific interactions with Csm5, a member of the type III effector complex, which facilitate nuclease recruitment/stimulation. Furthermore, we demonstrate that RNase R and PNPase are required to maintain robust anti-plasmid immunity, particularly when targeted transcripts are sparse. Altogether, our findings expand the known repertoire of accessory nucleases required for type III immunity and highlight the remarkable capacity of these systems to interface with diverse cellular pathways to ensure successful defense.
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Affiliation(s)
- Lucy Chou-Zheng
- Microbiology Department, University of Illinois Urbana-ChampaignUrbanaUnited States
| | - Asma Hatoum-Aslan
- Microbiology Department, University of Illinois Urbana-ChampaignUrbanaUnited States
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9
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Kolesnik MV, Fedorova I, Karneyeva KA, Artamonova DN, Severinov KV. Type III CRISPR-Cas Systems: Deciphering the Most Complex Prokaryotic Immune System. BIOCHEMISTRY. BIOKHIMIIA 2021; 86:1301-1314. [PMID: 34903162 PMCID: PMC8527444 DOI: 10.1134/s0006297921100114] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/01/2021] [Revised: 08/24/2021] [Accepted: 08/30/2021] [Indexed: 12/18/2022]
Abstract
The emergence and persistence of selfish genetic elements is an intrinsic feature of all living systems. Cellular organisms have evolved a plethora of elaborate defense systems that limit the spread of such genetic parasites. CRISPR-Cas are RNA-guided defense systems used by prokaryotes to recognize and destroy foreign nucleic acids. These systems acquire and store fragments of foreign nucleic acids and utilize the stored sequences as guides to recognize and destroy genetic invaders. CRISPR-Cas systems have been extensively studied, as some of them are used in various genome editing technologies. Although Type III CRISPR-Cas systems are among the most common CRISPR-Cas systems, they are also some of the least investigated ones, mostly due to the complexity of their action compared to other CRISPR-Cas system types. Type III effector complexes specifically recognize and cleave RNA molecules. The recognition of the target RNA activates the effector large subunit - the so-called CRISPR polymerase - which cleaves DNA and produces small cyclic oligonucleotides that act as signaling molecules to activate auxiliary effectors, notably non-specific RNases. In this review, we provide a historical overview of the sometimes meandering pathway of the Type III CRISPR research. We also review the current data on the structures and activities of Type III CRISPR-Cas systems components, their biological roles, and evolutionary history. Finally, using structural modeling with AlphaFold2, we show that the archaeal HRAMP signature protein, which heretofore has had no assigned function, is a degenerate relative of Type III CRISPR-Cas signature protein Cas10, suggesting that HRAMP systems have descended from Type III CRISPR-Cas systems or their ancestors.
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Affiliation(s)
- Matvey V Kolesnik
- Center of Life Science, Skolkovo Institute of Science and Technology, Moscow, 121205, Russia.
| | - Iana Fedorova
- Peter the Great St. Petersburg Polytechnic University, St. Petersburg, 195251, Russia.
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334, Russia
| | - Karyna A Karneyeva
- Center of Life Science, Skolkovo Institute of Science and Technology, Moscow, 121205, Russia.
| | - Daria N Artamonova
- Center of Life Science, Skolkovo Institute of Science and Technology, Moscow, 121205, Russia.
| | - Konstantin V Severinov
- Center of Life Science, Skolkovo Institute of Science and Technology, Moscow, 121205, Russia.
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334, Russia
- Waksman Institute of Microbiology, Piscataway, NJ 08854, USA
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10
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Lin J, Shen Y, Ni J, She Q. A type III-A CRISPR-Cas system mediates co-transcriptional DNA cleavage at the transcriptional bubbles in close proximity to active effectors. Nucleic Acids Res 2021; 49:7628-7643. [PMID: 34197611 PMCID: PMC8287949 DOI: 10.1093/nar/gkab590] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2021] [Revised: 06/18/2021] [Accepted: 06/24/2021] [Indexed: 12/26/2022] Open
Abstract
Many type III CRISPR–Cas systems rely on the cyclic oligoadenylate (cOA) signaling pathway to exert immunization. However, LdCsm, a type III-A lactobacilli immune system mediates efficient plasmid clearance in spite of lacking cOA signaling. Thus, the system provides a good model for detailed characterization of the RNA-activated DNase in vitro and in vivo. We found ATP functions as a ligand to enhance the LdCsm ssDNase, and the ATP enhancement is essential for in vivo plasmid clearance. In vitro assays demonstrated LdCsm cleaved transcriptional bubbles at any positions in non-template strand, suggesting that DNA cleavage may occur for transcribing DNA. Destiny of target plasmid versus nontarget plasmid in Escherichia coli cells was investigated, and this revealed that the LdCsm effectors mediated co-transcriptional DNA cleavage to both target and nontarget plasmids, suggesting LdCsm effectors can mediate DNA cleavage to any transcriptional bubbles in close proximity upon activation. Subcellular locations of active LdCsm effectors were then manipulated by differential expression of LdCsm and CTR, and the data supported the hypothesis. Strikingly, stepwise induction experiments indicated allowing diffusion of LdCsm effector led to massive chromosomal DNA degradation, suggesting this unique IIIA system can facilitate infection abortion to eliminate virus-infected cells.
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Affiliation(s)
- Jinzhong Lin
- CRISPR and Archaea Biology Research Center, State Key Laboratory of Microbial Technology, Shandong University, 72 Binhai Road, Jimo, Qingdao, Shandong 266237, P.R. China.,Archaea Centre, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark
| | - Yulong Shen
- CRISPR and Archaea Biology Research Center, State Key Laboratory of Microbial Technology, Shandong University, 72 Binhai Road, Jimo, Qingdao, Shandong 266237, P.R. China
| | - Jinfeng Ni
- CRISPR and Archaea Biology Research Center, State Key Laboratory of Microbial Technology, Shandong University, 72 Binhai Road, Jimo, Qingdao, Shandong 266237, P.R. China
| | - Qunxin She
- CRISPR and Archaea Biology Research Center, State Key Laboratory of Microbial Technology, Shandong University, 72 Binhai Road, Jimo, Qingdao, Shandong 266237, P.R. China.,Archaea Centre, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark
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11
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Athukoralage JS, White MF. Cyclic oligoadenylate signalling and regulation by ring nucleases during type III CRISPR defence. RNA (NEW YORK, N.Y.) 2021; 27:rna.078739.121. [PMID: 33986148 PMCID: PMC8284326 DOI: 10.1261/rna.078739.121] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/05/2021] [Accepted: 05/08/2021] [Indexed: 06/12/2023]
Abstract
In prokaryotes, CRISPR-Cas immune systems recognise and cleave foreign nucleic acids to defend against Mobile Genetic Elements (MGEs). Type III CRISPR-Cas complexes also synthesise cyclic oligoadenylate (cOA) second messengers, which activate CRISPR ancillary proteins involved in antiviral defence. In particular, cOA-stimulated nucleases degrade RNA and DNA non-specifically, which slows MGE replication but also impedes cell growth, necessitating mechanisms to eliminate cOA in order to mitigate collateral damage. Extant cOA is degraded by a new class of enzyme termed a 'ring nuclease', which cleaves cOA specifically and switches off CRISPR ancillary enzymes. Several ring nuclease families have been characterised to date, including a family used by MGEs to circumvent CRISPR immunity, and encompass diverse protein folds and distinct cOA cleavage mechanisms. In this review we outline cOA signalling, discuss how different ring nucleases regulate the cOA signalling pathway, and reflect on parallels between cyclic nucleotide-based immune systems to reveal new areas for exploration.
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12
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Riediger M, Spät P, Bilger R, Voigt K, Maček B, Hess WR. Analysis of a photosynthetic cyanobacterium rich in internal membrane systems via gradient profiling by sequencing (Grad-seq). THE PLANT CELL 2021; 33:248-269. [PMID: 33793824 PMCID: PMC8136920 DOI: 10.1093/plcell/koaa017] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/10/2020] [Accepted: 11/12/2020] [Indexed: 05/23/2023]
Abstract
Although regulatory small RNAs have been reported in photosynthetic cyanobacteria, the lack of clear RNA chaperones involved in their regulation poses a conundrum. Here, we analyzed the full complement of cellular RNAs and proteins using gradient profiling by sequencing (Grad-seq) in Synechocystis 6803. Complexes with overlapping subunits such as the CpcG1-type versus the CpcL-type phycobilisomes or the PsaK1 versus PsaK2 photosystem I pre(complexes) could be distinguished, supporting the high quality of this approach. Clustering of the in-gradient distribution profiles followed by several additional criteria yielded a short list of potential RNA chaperones that include an YlxR homolog and a cyanobacterial homolog of the KhpA/B complex. The data suggest previously undetected complexes between accessory proteins and CRISPR-Cas systems, such as a Csx1-Csm6 ribonucleolytic defense complex. Moreover, the exclusive association of either RpoZ or 6S RNA with the core RNA polymerase complex and the existence of a reservoir of inactive sigma-antisigma complexes is suggested. The Synechocystis Grad-seq resource is available online at https://sunshine.biologie.uni-freiburg.de/GradSeqExplorer/ providing a comprehensive resource for the functional assignment of RNA-protein complexes and multisubunit protein complexes in a photosynthetic organism.
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Affiliation(s)
- Matthias Riediger
- Genetics and Experimental Bioinformatics, Faculty of Biology, University of Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany
| | - Philipp Spät
- Department of Quantitative Proteomics, Interfaculty Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany
| | - Raphael Bilger
- Genetics and Experimental Bioinformatics, Faculty of Biology, University of Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany
| | - Karsten Voigt
- IT Administration, Institute of Biology 3, Faculty of Biology, University of Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany
| | - Boris Maček
- Department of Quantitative Proteomics, Interfaculty Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany
| | - Wolfgang R Hess
- Genetics and Experimental Bioinformatics, Faculty of Biology, University of Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany
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13
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Expression, purification, and characterization of a membrane-associated cyclic oligo-adenylate degrader from Sulfolobus islandicus. STAR Protoc 2021; 2:100299. [PMID: 33537681 PMCID: PMC7841402 DOI: 10.1016/j.xpro.2021.100299] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Type III CRISPR-cas systems initiate cyclic oligo-adenylate (cOA) signaling to initiate immune response. Previously, we identified that a membrane-associated DHH-DHHA1 family protein from Sulfolobus islandicus efficiently degrades cOA. Here, we provide detailed protocols for expression and purification of the protein from its native host and a cOA degradation assay with the purified enzyme. The methodology should be of interest for researchers studying Sulfolobus, membrane-associated proteins, or type III CRISPR-cas systems. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020).
Construct a Sulfolobus strain to express a membrane-associated DHH-DHHA1 protein (MAD) Purify MAD by detergent treatment followed by chromatography Analyze the degradation of type III CRISPR second messenger by MAD
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14
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Huang F, Zhu B. The Cyclic Oligoadenylate Signaling Pathway of Type III CRISPR-Cas Systems. Front Microbiol 2021; 11:602789. [PMID: 33552016 PMCID: PMC7854544 DOI: 10.3389/fmicb.2020.602789] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2020] [Accepted: 11/11/2020] [Indexed: 12/24/2022] Open
Abstract
Type III CRISPR-Cas systems, which are widespread in both bacteria and archaea, provide immunity against DNA viruses and plasmids in a transcription-dependent manner. Since an unprecedented cyclic oligoadenylate (cOA) signaling pathway was discovered in type III systems in 2017, the cOA signaling has been extensively studied in recent 3 years, which has expanded our understanding of type III systems immune defense and also its counteraction by viruses. In this review, we summarized recent advances in cOA synthesis, cOA-activated effector protein, cOA signaling-mediated immunoprotection, and cOA signaling inhibition, and highlighted the crosstalk between cOA signaling and other cyclic oligonucleotide-mediated immunity discovered very recently.
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Affiliation(s)
- Fengtao Huang
- Key Laboratory of Molecular Biophysics, the Ministry of Education, College of Life Science and Technology and Shenzhen College, Huazhong University of Science and Technology, Wuhan, China
| | - Bin Zhu
- Key Laboratory of Molecular Biophysics, the Ministry of Education, College of Life Science and Technology and Shenzhen College, Huazhong University of Science and Technology, Wuhan, China
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15
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Shivram H, Cress BF, Knott GJ, Doudna JA. Controlling and enhancing CRISPR systems. Nat Chem Biol 2021; 17:10-19. [PMID: 33328654 PMCID: PMC8101458 DOI: 10.1038/s41589-020-00700-7] [Citation(s) in RCA: 100] [Impact Index Per Article: 25.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2020] [Accepted: 10/22/2020] [Indexed: 12/17/2022]
Abstract
Many bacterial and archaeal organisms use clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) systems to defend themselves from mobile genetic elements. These CRISPR-Cas systems are classified into six types based on their composition and mechanism. CRISPR-Cas enzymes are widely used for genome editing and offer immense therapeutic opportunity to treat genetic diseases. To realize their full potential, it is important to control the timing, duration, efficiency and specificity of CRISPR-Cas enzyme activities. In this Review we discuss the mechanisms of natural CRISPR-Cas regulatory biomolecules and engineering strategies that enhance or inhibit CRISPR-Cas immunity by altering enzyme function. We also discuss the potential applications of these CRISPR regulators and highlight unanswered questions about their evolution and purpose in nature.
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Affiliation(s)
- Haridha Shivram
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA
- Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA
| | - Brady F Cress
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA
- Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA
| | - Gavin J Knott
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA
- Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA
- Monash Biomedicine Discovery Institute, Department of Biochemistry & Molecular Biology, Monash University, Victoria, Australia
| | - Jennifer A Doudna
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
- Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
- Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA.
- California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA, USA.
- Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA, USA.
- MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, Berkeley, CA, USA.
- Gladstone Institutes, University of California, San Francisco, San Francisco, CA, USA.
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16
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Zink IA, Wimmer E, Schleper C. Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales. Biomolecules 2020; 10:E1523. [PMID: 33172134 PMCID: PMC7694759 DOI: 10.3390/biom10111523] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2020] [Revised: 10/31/2020] [Accepted: 11/03/2020] [Indexed: 12/13/2022] Open
Abstract
Prokaryotes are constantly coping with attacks by viruses in their natural environments and therefore have evolved an impressive array of defense systems. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system found in the majority of archaea and about half of bacteria which stores pieces of infecting viral DNA as spacers in genomic CRISPR arrays to reuse them for specific virus destruction upon a second wave of infection. In detail, small CRISPR RNAs (crRNAs) are transcribed from CRISPR arrays and incorporated into type-specific CRISPR effector complexes which further degrade foreign nucleic acids complementary to the crRNA. This review gives an overview of CRISPR immunity to newcomers in the field and an update on CRISPR literature in archaea by comparing the functional mechanisms and abundances of the diverse CRISPR types. A bigger fraction is dedicated to the versatile and prevalent CRISPR type III systems, as tremendous progress has been made recently using archaeal models in discerning the controlled molecular mechanisms of their unique tripartite mode of action including RNA interference, DNA interference and the unique cyclic-oligoadenylate signaling that induces promiscuous RNA shredding by CARF-domain ribonucleases. The second half of the review spotlights CRISPR in archaea outlining seminal in vivo and in vitro studies in model organisms of the euryarchaeal and crenarchaeal phyla, including the application of CRISPR-Cas for genome editing and gene silencing. In the last section, a special focus is laid on members of the crenarchaeal hyperthermophilic order Sulfolobales by presenting a thorough comparative analysis about the distribution and abundance of CRISPR-Cas systems, including arrays and spacers as well as CRISPR-accessory proteins in all 53 genomes available to date. Interestingly, we find that CRISPR type III and the DNA-degrading CRISPR type I complexes co-exist in more than two thirds of these genomes. Furthermore, we identified ring nuclease candidates in all but two genomes and found that they generally co-exist with the above-mentioned CARF domain ribonucleases Csx1/Csm6. These observations, together with published literature allowed us to draft a working model of how CRISPR-Cas systems and accessory proteins cross talk to establish native CRISPR anti-virus immunity in a Sulfolobales cell.
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17
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Smalakyte D, Kazlauskiene M, F Havelund J, Rukšėnaitė A, Rimaite A, Tamulaitiene G, Færgeman NJ, Tamulaitis G, Siksnys V. Type III-A CRISPR-associated protein Csm6 degrades cyclic hexa-adenylate activator using both CARF and HEPN domains. Nucleic Acids Res 2020; 48:9204-9217. [PMID: 32766806 PMCID: PMC7498309 DOI: 10.1093/nar/gkaa634] [Citation(s) in RCA: 41] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2020] [Revised: 07/12/2020] [Accepted: 08/06/2020] [Indexed: 12/26/2022] Open
Abstract
The type III CRISPR–Cas systems provide immunity against invading nucleic acids through the coordinated transcription-dependent DNA targeting and cyclic adenylate (cAn)-activated RNA degradation. Here, we show that both these pathways contribute to the Streptococcus thermophilus (St) type III-A CRISPR–Cas immunity. HPLC-MS analysis revealed that in the heterologous Escherichia coli host the StCsm effector complex predominantly produces cA5 and cA6. cA6 acts as a signaling molecule that binds to the CARF domain of StCsm6 to activate non-specific RNA degradation by the HEPN domain. By dissecting StCsm6 domains we demonstrate that both CARF and HEPN domains act as ring nucleases that degrade cAns to switch signaling off. CARF ring nuclease converts cA6 to linear A6>p and to the final A3>p product. HEPN domain, which typically degrades RNA, also shows ring nuclease activity and indiscriminately degrades cA6 or other cAns down to A>p. We propose that concerted action of both ring nucleases enables self-regulation of the RNase activity in the HEPN domain and eliminates all cAn secondary messengers in the cell when viral infection is combated by a coordinated action of Csm effector and the cA6-activated Csm6 ribonuclease.
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Affiliation(s)
- Dalia Smalakyte
- Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania
| | - Migle Kazlauskiene
- Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania
| | - Jesper F Havelund
- Department of Biochemistry and Molecular Biology, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark
| | - Audronė Rukšėnaitė
- Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania
| | - Auguste Rimaite
- Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania
| | - Giedre Tamulaitiene
- Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania
| | - Nils J Færgeman
- Department of Biochemistry and Molecular Biology, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark
| | - Gintautas Tamulaitis
- Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania
| | - Virginijus Siksnys
- Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania
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18
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Liu TY, Doudna JA. Chemistry of Class 1 CRISPR-Cas effectors: Binding, editing, and regulation. J Biol Chem 2020; 295:14473-14487. [PMID: 32817336 PMCID: PMC7573268 DOI: 10.1074/jbc.rev120.007034] [Citation(s) in RCA: 65] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2020] [Revised: 08/13/2020] [Indexed: 12/19/2022] Open
Abstract
Among the multiple antiviral defense mechanisms found in prokaryotes, CRISPR-Cas systems stand out as the only known RNA-programmed pathways for detecting and destroying bacteriophages and plasmids. Class 1 CRISPR-Cas systems, the most widespread and diverse of these adaptive immune systems, use an RNA-guided multiprotein complex to find foreign nucleic acids and trigger their destruction. In this review, we describe how these multisubunit complexes target and cleave DNA and RNA and how regulatory molecules control their activities. We also highlight similarities to and differences from Class 2 CRISPR-Cas systems, which use a single-protein effector, as well as other types of bacterial and eukaryotic immune systems. We summarize current applications of the Class 1 CRISPR-Cas systems for DNA/RNA modification, control of gene expression, and nucleic acid detection.
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Affiliation(s)
- Tina Y Liu
- Department of Molecular and Cell Biology, University of California, Berkeley, California, USA
| | - Jennifer A Doudna
- Department of Molecular and Cell Biology, University of California, Berkeley, California, USA
- Department of Chemistry, University of California, Berkeley, California, USA
- California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, California, USA
- Innovative Genomics Institute, University of California, Berkeley, California, USA
- Howard Hughes Medical Institute, University of California, Berkeley, California, USA
- MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA
- Gladstone Institutes, University of California, San Francisco, California, USA
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19
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Ye Q, Zhao X, Liu J, Zeng Z, Zhang Z, Liu T, Li Y, Han W, Peng N. CRISPR-Associated Factor Csa3b Regulates CRISPR Adaptation and Cmr-Mediated RNA Interference in Sulfolobus islandicus. Front Microbiol 2020; 11:2038. [PMID: 32983033 PMCID: PMC7480081 DOI: 10.3389/fmicb.2020.02038] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2020] [Accepted: 07/31/2020] [Indexed: 12/14/2022] Open
Abstract
Acquisition of spacers confers the CRISPR–Cas system with the memory to defend against invading mobile genetic elements. We previously reported that the CRISPR-associated factor Csa3a triggers CRISPR adaptation in Sulfolobus islandicus. However, a feedback regulation of CRISPR adaptation remains unclear. Here we show that another CRISPR-associated factor, Csa3b, binds a cyclic oligoadenylate (cOA) analog (5′-CAAAA-3′) and mutation at its CARF domain, which reduces the binding affinity. Csa3b also binds the promoter of adaptation cas genes, and the cOA analog enhances their binding probably by allosteric regulation. Deletion of the csa3b gene triggers spacer acquisition from both plasmid and viral DNAs, indicating that Csa3b acted as a repressor for CRISPR adaptation. Moreover, we also find that Csa3b activates the expression of subtype cmr-α and cmr-β genes according to transcriptome data and demonstrate that Csa3b binds the promoters of cmr genes. The deletion of the csa3b gene reduces Cmr-mediated RNA interference activity, indicating that Csa3b acts as a transcriptional activator for Cmr-mediated RNA interference. In summary, our findings reveal a novel pathway for the regulation of CRISPR adaptation and CRISPR–Cmr RNA interference in S. islandicus. Our results also suggest a feedback repression of CRIPSR adaptation by the Csa3b factor and the cOA signal produced by the Cmr complex at the CRISPR interference stage.
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Affiliation(s)
- Qing Ye
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China
| | - Xueqiao Zhao
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China
| | - Jilin Liu
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China
| | - Zhifeng Zeng
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China
| | - Zhufeng Zhang
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China
| | - Tao Liu
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China
| | - Yingjun Li
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China
| | - Wenyuan Han
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China
| | - Nan Peng
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China
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20
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Lin J, Feng M, Zhang H, She Q. Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase. Cell Discov 2020; 6:29. [PMID: 32411384 PMCID: PMC7214462 DOI: 10.1038/s41421-020-0160-4] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2019] [Accepted: 03/19/2020] [Indexed: 02/08/2023] Open
Abstract
Antiviral defense by type III CRISPR-Cas systems relies on two distinct activities of their effectors: the RNA-activated DNA cleavage and synthesis of cyclic oligoadenylate. Both activities are featured as indiscriminate nucleic acid cleavage and subjected to the spatiotemporal regulation. To yield further insights into the involved mechanisms, we reconstituted LdCsm, a lactobacilli III-A system in Escherichia coli. Upon activation by target RNA, this immune system mediates robust DNA degradation but lacks the synthesis of cyclic oligoadenylates. Mutagenesis of the Csm3 and Cas10 conserved residues revealed that Csm3 and multiple structural domains in Cas10 function in the allosteric regulation to yield an active enzyme. Target RNAs carrying various truncations in the 3' anti-tag were designed and tested for their influence on DNA binding and DNA cleavage of LdCsm. Three distinct states of ternary LdCsm complexes were identified. In particular, binding of target RNAs carrying a single nucleotide in the 3' anti-tag to LdCsm yielded an active LdCsm DNase regardless whether the nucleotide shows a mismatch, as in the cognate target RNA (CTR), or a match, as in the noncognate target RNA (NTR), to the 5' tag of crRNA. In addition, further increasing the number of 3' anti-tag in CTR facilitated the substrate binding and enhanced the substrate degradation whereas doing the same as in NTR gradually decreased the substrate binding and eventually shut off the DNA cleavage by the enzyme. Together, these results provide the mechanistic insights into the allosteric activation and repression of LdCsm enzymes.
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Affiliation(s)
- Jinzhong Lin
- Archaea Centre, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark
| | - Mingxia Feng
- Archaea Centre, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark
- College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China
| | - Heping Zhang
- Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, 010018 Hohhot, China
| | - Qunxin She
- Microbial Technology Institute and State Key Laboratory of Microbial Technology, Shandong University, 72 Binhai Road, Jimo, 266237 Qingdao, Shandong China
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21
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Foster K, Grüschow S, Bailey S, White MF, Terns MP. Regulation of the RNA and DNA nuclease activities required for Pyrococcus furiosus Type III-B CRISPR-Cas immunity. Nucleic Acids Res 2020; 48:4418-4434. [PMID: 32198888 PMCID: PMC7192623 DOI: 10.1093/nar/gkaa176] [Citation(s) in RCA: 33] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2019] [Revised: 02/28/2020] [Accepted: 03/19/2020] [Indexed: 12/25/2022] Open
Abstract
Type III CRISPR-Cas prokaryotic immune systems provide anti-viral and anti-plasmid immunity via a dual mechanism of RNA and DNA destruction. Upon target RNA interaction, Type III crRNP effector complexes become activated to cleave both target RNA (via Cas7) and target DNA (via Cas10). Moreover, trans-acting endoribonucleases, Csx1 or Csm6, can promote the Type III immune response by destroying both invader and host RNAs. Here, we characterize how the RNase and DNase activities associated with Type III-B immunity in Pyrococcus furiosus (Pfu) are regulated by target RNA features and second messenger signaling events. In vivo mutational analyses reveal that either the DNase activity of Cas10 or the RNase activity of Csx1 can effectively direct successful anti-plasmid immunity. Biochemical analyses confirmed that the Cas10 Palm domains convert ATP into cyclic oligoadenylate (cOA) compounds that activate the ribonuclease activity of Pfu Csx1. Furthermore, we show that the HEPN domain of the adenosine-specific endoribonuclease, Pfu Csx1, degrades cOA signaling molecules to provide an auto-inhibitory off-switch of Csx1 activation. Activation of both the DNase and cOA generation activities require target RNA binding and recognition of distinct target RNA 3' protospacer flanking sequences. Our results highlight the complex regulatory mechanisms controlling Type III CRISPR immunity.
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Affiliation(s)
- Kawanda Foster
- Department of Microbiology, University of Georgia, Athens, GA 30602, USA
| | - Sabine Grüschow
- Biomedical Sciences Research Complex, School of Biology, University of St Andrews, St Andrews KY16 9ST, UK
| | - Scott Bailey
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Malcolm F White
- Biomedical Sciences Research Complex, School of Biology, University of St Andrews, St Andrews KY16 9ST, UK
| | - Michael P Terns
- Department of Microbiology, University of Georgia, Athens, GA 30602, USA
- Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA
- Department of Genetics, University of Georgia, Athens, GA 30602, USA
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22
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Structure and Mechanism of a Cyclic Trinucleotide-Activated Bacterial Endonuclease Mediating Bacteriophage Immunity. Mol Cell 2020; 77:723-733.e6. [PMID: 31932164 DOI: 10.1016/j.molcel.2019.12.010] [Citation(s) in RCA: 134] [Impact Index Per Article: 26.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2019] [Revised: 10/04/2019] [Accepted: 12/12/2019] [Indexed: 12/26/2022]
Abstract
Bacteria possess an array of defenses against foreign invaders, including a broadly distributed bacteriophage defense system termed CBASS (cyclic oligonucleotide-based anti-phage signaling system). In CBASS systems, a cGAS/DncV-like nucleotidyltransferase synthesizes cyclic di- or tri-nucleotide second messengers in response to infection, and these molecules activate diverse effectors to mediate bacteriophage immunity via abortive infection. Here, we show that the CBASS effector NucC is related to restriction enzymes but uniquely assembles into a homotrimer. Binding of NucC trimers to a cyclic tri-adenylate second messenger promotes assembly of a NucC homohexamer competent for non-specific double-strand DNA cleavage. In infected cells, NucC activation leads to complete destruction of the bacterial chromosome, causing cell death prior to completion of phage replication. In addition to CBASS systems, we identify NucC homologs in over 30 type III CRISPR/Cas systems, where they likely function as accessory nucleases activated by cyclic oligoadenylate second messengers synthesized by these systems' effector complexes.
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23
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Grüschow S, Athukoralage JS, Graham S, Hoogeboom T, White MF. Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence. Nucleic Acids Res 2019; 47:9259-9270. [PMID: 31392987 PMCID: PMC6755085 DOI: 10.1093/nar/gkz676] [Citation(s) in RCA: 45] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2019] [Revised: 07/19/2019] [Accepted: 07/30/2019] [Indexed: 12/26/2022] Open
Abstract
The CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogen Mycobacterium tuberculosis. Here, we report a comprehensive analysis of the in vitro and in vivo activities of the type III-A M. tuberculosis CRISPR system. We demonstrate that immunity against MGE may be achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show for the first time that a type III CRISPR system can be reprogrammed by replacing the effector protein, which may be relevant for maintenance of immunity in response to pressure from viral anti-CRISPRs. These observations demonstrate that M. tuberculosis has a fully-functioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.
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Affiliation(s)
- Sabine Grüschow
- Biomedical Sciences Research Complex, School of Biology, University of St Andrews, St Andrews KY16 9ST, UK
| | - Januka S Athukoralage
- Biomedical Sciences Research Complex, School of Biology, University of St Andrews, St Andrews KY16 9ST, UK
| | - Shirley Graham
- Biomedical Sciences Research Complex, School of Biology, University of St Andrews, St Andrews KY16 9ST, UK
| | - Tess Hoogeboom
- Biomedical Sciences Research Complex, School of Biology, University of St Andrews, St Andrews KY16 9ST, UK
| | - Malcolm F White
- Biomedical Sciences Research Complex, School of Biology, University of St Andrews, St Andrews KY16 9ST, UK
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24
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Li Y, Peng N. Endogenous CRISPR-Cas System-Based Genome Editing and Antimicrobials: Review and Prospects. Front Microbiol 2019; 10:2471. [PMID: 31708910 PMCID: PMC6824031 DOI: 10.3389/fmicb.2019.02471] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2019] [Accepted: 10/15/2019] [Indexed: 12/17/2022] Open
Abstract
CRISPR-Cas systems adapt “memories” via spacers from viruses and plasmids to develop adaptive immunity against mobile genetic elements. Mature CRISPR RNAs guide CRISPR-associated nucleases to site-specifically cleave target DNA or RNA, providing an efficient genome engineering tool for organisms of all three kingdoms. Cas9, Cas12, and Cas13 are single proteins with multiple domains that are the most widely used CRISPR nucleases of the Class 2 system. However, these CRISPR endonucleases are large in size, leading to difficulty for manipulation and toxicity for cells. Most archaeal genomes and half of the bacterial genomes encode different types of CRISPR-Cas systems. Therefore, developing endogenous CRISPR-Cas systems-based genome editing will simplify manipulations and increase editing efficiency in prokaryotic cells. Here, we review the current applications and discuss the prospects of using endogenous CRISPR nucleases for genome engineering and CRISPR-based antimicrobials.
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Affiliation(s)
- Yingjun Li
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China
| | - Nan Peng
- State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China
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Structure of Csx1-cOA 4 complex reveals the basis of RNA decay in Type III-B CRISPR-Cas. Nat Commun 2019; 10:4302. [PMID: 31541109 PMCID: PMC6754442 DOI: 10.1038/s41467-019-12244-z] [Citation(s) in RCA: 61] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2019] [Accepted: 08/29/2019] [Indexed: 12/26/2022] Open
Abstract
Type III CRISPR-Cas multisubunit complexes cleave ssRNA and ssDNA. These activities promote the generation of cyclic oligoadenylate (cOA), which activates associated CRISPR-Cas RNases from the Csm/Csx families, triggering a massive RNA decay to provide immunity from genetic invaders. Here we present the structure of Sulfolobus islandicus (Sis) Csx1-cOA4 complex revealing the allosteric activation of its RNase activity. SisCsx1 is a hexamer built by a trimer of dimers. Each dimer forms a cOA4 binding site and a ssRNA catalytic pocket. cOA4 undergoes a conformational change upon binding in the second messenger binding site activating ssRNA degradation in the catalytic pockets. Activation is transmitted in an allosteric manner through an intermediate HTH domain, which joins the cOA4 and catalytic sites. The RNase functions in a sequential cooperative fashion, hydrolyzing phosphodiester bonds in 5′-C-C-3′. The degradation of cOA4 by Ring nucleases deactivates SisCsx1, suggesting that this enzyme could be employed in biotechnological applications. Type III CRISPR-Cas RNases from the Csm and Csx families are activated by cyclic oligoadenylates (cOA). Here the authors present the cOA bound Sulfolobus islandicus Csx1 structure, which forms a hexamer and reveal an allosteric mechanism for the activation of Csx1 RNase.
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Second Messenger cA 4 Formation within the Composite Csm1 Palm Pocket of Type III-A CRISPR-Cas Csm Complex and Its Release Path. Mol Cell 2019; 75:933-943.e6. [PMID: 31326272 DOI: 10.1016/j.molcel.2019.06.013] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2019] [Revised: 05/09/2019] [Accepted: 06/09/2019] [Indexed: 12/26/2022]
Abstract
Target RNA binding to crRNA-bound type III-A CRISPR-Cas multi-subunit Csm surveillance complexes activates cyclic-oligoadenylate (cAn) formation from ATP subunits positioned within the composite pair of Palm domain pockets of the Csm1 subunit. The generated cAn second messenger in turn targets the CARF domain of trans-acting RNase Csm6, triggering its HEPN domain-based RNase activity. We have undertaken cryo-EM studies on multi-subunit Thermococcus onnurineus Csm effector ternary complexes, as well as X-ray studies on Csm1-Csm4 cassette, both bound to substrate (AMPPNP), intermediates (pppAn), and products (cAn), to decipher mechanistic aspects of cAn formation and release. A network of intermolecular hydrogen bond alignments accounts for the observed adenosine specificity, with ligand positioning dictating formation of linear pppAn intermediates and subsequent cAn formation by cyclization. We combine our structural results with published functional studies to highlight mechanistic insights into the role of the Csm effector complex in mediating the cAn signaling pathway.
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Guo T, Zheng F, Zeng Z, Yang Y, Li Q, She Q, Han W. Cmr3 regulates the suppression on cyclic oligoadenylate synthesis by tag complementarity in a Type III-B CRISPR-Cas system. RNA Biol 2019; 16:1513-1520. [PMID: 31298604 DOI: 10.1080/15476286.2019.1642725] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Type III CRISPR-Cas systems code for a multi-subunit ribonucleoprotein (RNP) complex that mediates DNA cleavage and synthesizes cyclic oligoadenylate (cOA) second messenger to confer anti-viral immunity. Both immune activities are to be activated upon binding to target RNA transcripts by their complementarity to crRNA, and autoimmunity avoidance is determined by extended complementarity between the 5'-repeat tag of crRNA and 3'-flanking sequences of target transcripts (anti-tag). However, as to how the strategy could achieve stringent autoimmunity avoidance remained elusive. In this study, we systematically investigated how the complementarity of the crRNA 5'-tag and anti-tag (i.e., tag complementarity) could affect the interference activities (DNA cleavage activity and cOA synthesis activity) of Cmr-α, a type III-B system in Sulfolobus islandicus Rey15A. The results revealed an increasing suppression on both activities by increasing degrees of tag complementarity and a critical function of the 7th nucleotide of crRNA in avoiding autoimmunity. More importantly, mutagenesis of Cmr3α exerts either positive or negative effects on the cOA synthesis activity depending on the degrees of tag complementarity, suggesting that the subunit, coupling with the interaction between crRNA tag and anti-tag, function in facilitating immunity and avoiding autoimmunity in Type III-B systems.
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Affiliation(s)
- Tong Guo
- Danish Archaea Center, Department of Biology, University of Copenhagen , Copenhagen N , Denmark
| | - Fan Zheng
- State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University , Wuhan , China
| | - Zhifeng Zeng
- State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University , Wuhan , China
| | - Yang Yang
- State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University , Wuhan , China
| | - Qi Li
- State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University , Wuhan , China
| | - Qunxin She
- Danish Archaea Center, Department of Biology, University of Copenhagen , Copenhagen N , Denmark.,State Key Laboratory of Microbial Technology, Shandong University , Qingdao , China
| | - Wenyuan Han
- State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University , Wuhan , China
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Varble A, Marraffini LA. Three New Cs for CRISPR: Collateral, Communicate, Cooperate. Trends Genet 2019; 35:446-456. [PMID: 31036344 PMCID: PMC6525018 DOI: 10.1016/j.tig.2019.03.009] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2019] [Revised: 03/28/2019] [Accepted: 03/28/2019] [Indexed: 12/18/2022]
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated (cas) genes provide protection against invading phages and plasmids in prokaryotes. Typically, short sequences are captured from the genome of the invader, integrated into the CRISPR locus, and transcribed into short RNAs that direct RNA-guided Cas nucleases to the nucleic acids of the invader for their degradation. Recent work in the field has revealed unexpected features of the CRISPR-Cas mechanism: (i) collateral, nonspecific, cleavage of host nucleic acids; (ii) secondary messengers that amplify the immune response; and (iii) immunosuppression of CRISPR targeting by phage-encoded inhibitors. Here, we review these new and exciting findings.
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Affiliation(s)
- Andrew Varble
- Laboratory of Bacteriology, The Rockefeller University, 1230 York Ave, New York, NY 10065, USA.
| | - Luciano A Marraffini
- Laboratory of Bacteriology, The Rockefeller University, 1230 York Ave, New York, NY 10065, USA; Howard Hughes Medical Institute, The Rockefeller University, 1230 York Ave, New York, NY 10065, USA.
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Pan S, Li Q, Deng L, Jiang S, Jin X, Peng N, Liang Y, She Q, Li Y. A seed motif for target RNA capture enables efficient immune defence by a type III-B CRISPR-Cas system. RNA Biol 2019; 16:1166-1178. [PMID: 31096876 DOI: 10.1080/15476286.2019.1618693] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
CRISPR-Cas systems provide an adaptive defence against foreign nucleic acids guided by small RNAs (crRNAs) in archaea and bacteria. The Type III CRISPR systems are reported to carry RNase, RNA-activated DNase and cyclic oligoadenylate (cOA) synthetase activity, and are significantly different from other CRISPR systems. However, detailed features of target recognition, which are essential for enhancing target specificity remain unknown in Type III CRISPR systems. Here, we show that the Type III-B Cmr-α system in S. islandicus generates two constant lengths of crRNA independent of the length of the spacer. Either mutation at the 3'-end of crRNA or target truncation greatly influences the target capture and cleavage by the Cmr-α effector complex. Furthermore, we found that cleavage at the tag-proximal site on the target RNA by the Cmr-α RNP complex is delayed relative to the other sites, which probably provides Cas10 more time to function as a guard against invaders. Using a mutagenesis assay in vivo, we discovered that a seed motif located at the tag-distal region of the crRNA is required by Cmr1α for target RNA capture by the Cmr-α system thereby enhancing target specificity and efficiency. These findings further refine the model for immune defence of Type III-B CRISPR-Cas system, commencing on capture, cleavage and regulation.
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Affiliation(s)
- Saifu Pan
- a State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University , Wuhan , China
| | - Qi Li
- a State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University , Wuhan , China
| | - Ling Deng
- b Archaea Centre, Department of Biology, University of Copenhagen , Copenhagen , Denmark
| | - Suping Jiang
- a State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University , Wuhan , China
| | - Xuexia Jin
- a State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University , Wuhan , China
| | - Nan Peng
- a State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University , Wuhan , China
| | - Yunxiang Liang
- a State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University , Wuhan , China
| | - Qunxin She
- a State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University , Wuhan , China.,b Archaea Centre, Department of Biology, University of Copenhagen , Copenhagen , Denmark
| | - Yingjun Li
- a State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University , Wuhan , China
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Johnson K, Learn BA, Estrella MA, Bailey S. Target sequence requirements of a type III-B CRISPR-Cas immune system. J Biol Chem 2019; 294:10290-10299. [PMID: 31110048 DOI: 10.1074/jbc.ra119.008728] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2019] [Revised: 05/07/2019] [Indexed: 12/26/2022] Open
Abstract
CRISPR-Cas systems are RNA-based immune systems that protect many prokaryotes from invasion by viruses and plasmids. Type III CRISPR systems are unique, as their targeting mechanism requires target transcription. Upon transcript binding, DNA cleavage by type III effector complexes is activated. Type III systems must differentiate between invader and native transcripts to prevent autoimmunity. Transcript origin is dictated by the sequence that flanks the 3' end of the RNA target site (called the PFS). However, how the PFS is recognized may vary among different type III systems. Here, using purified proteins and in vitro assays, we define how the type III-B effector from the hyperthermophilic bacterium Thermotoga maritima discriminates between native and invader transcripts. We show that native transcripts are recognized by base pairing at positions -2 to -5 of the PFS and by a guanine at position -1, which is not recognized by base pairing. We also show that mismatches with the RNA target are highly tolerated in this system, except for those nucleotides adjacent to the PFS. These findings define the target requirement for the type III-B system from T. maritima and provide a framework for understanding the target requirements of type III systems as a whole.
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Affiliation(s)
- Kaitlin Johnson
- From the Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health and
| | - Brian A Learn
- From the Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health and
| | - Michael A Estrella
- From the Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health and
| | - Scott Bailey
- From the Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health and .,Department of Biophysics and Biophysical Chemistry, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205
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A Type III CRISPR Ancillary Ribonuclease Degrades Its Cyclic Oligoadenylate Activator. J Mol Biol 2019; 431:2894-2899. [PMID: 31071326 PMCID: PMC6599890 DOI: 10.1016/j.jmb.2019.04.041] [Citation(s) in RCA: 58] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2019] [Revised: 04/22/2019] [Accepted: 04/29/2019] [Indexed: 12/26/2022]
Abstract
Cyclic oligoadenylate (cOA) secondary messengers are generated by type III CRISPR systems in response to viral infection. cOA allosterically activates the CRISPR ancillary ribonucleases Csx1/Csm6, which degrade RNA non-specifically using a HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) active site. This provides effective immunity but can also lead to growth arrest in infected cells, necessitating a means to deactivate the ribonuclease once viral infection has been cleared. In the crenarchaea, dedicated ring nucleases degrade cA4 (cOA consisting of 4 AMP units), but the equivalent enzyme has not been identified in bacteria. We demonstrate that, in Thermus thermophilus HB8, the uncharacterized protein TTHB144 is a cA4-activated HEPN ribonuclease that also degrades its activator. TTHB144 binds and degrades cA4 at an N-terminal CARF (CRISPR-associated Rossman fold) domain. The two activities can be separated by site-directed mutagenesis. TTHB144 is thus the first example of a self-limiting CRISPR ribonuclease.
TTHB144 is a cyclic tetra-adenylate activated ribonuclease. TTHB144 degrades cyclic tetra-adenylate at its CARF domain. Self-limiting enzymes like TTHB144 may regulate anti-viral signaling in bacteria.
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Molecular mechanisms of III-B CRISPR–Cas systems in archaea. Emerg Top Life Sci 2018; 2:483-491. [DOI: 10.1042/etls20180023] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2018] [Revised: 08/26/2018] [Accepted: 08/31/2018] [Indexed: 12/26/2022]
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems provide the adaptive antiviral immunity against invasive genetic elements in archaea and bacteria. These immune systems are divided into at least six different types, among which Type III CRISPR–Cas systems show several distinct antiviral activities as demonstrated from the investigation of bacterial III-A and archaeal III-B systems in the past decade. First, although initial experiments suggested that III-A systems provided DNA interference activity, whereas III-B system was active only in RNA interference, these immune systems were subsequently found to mediate the transcription-dependent DNA interference and the dual DNA/RNA interference. Second, their ribonucleoprotein (RNP) complexes show target RNA (tgRNA) cleavage by a ruler mechanism and RNA-activated indiscriminate single-stranded DNA cleavage, the latter of which is subjected to spatiotemporal regulation such that the DNase activity occurs only at the right place in the right time. Third, RNPs of Type III systems catalyse the synthesis of cyclic oligoadenylates (cOAs) that function as second messengers to activate Csm6 and Csx1, both of which are potent Cas accessory RNases after activation. To date, Type III CRISPR systems are the only known antiviral immunity that utilizes multiple interference mechanisms for antiviral defence.
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