Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    We will mail you new results for this query: (q=crispr AND metisnummer==1014585)
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Snel en simpel genen repareren: opmars DNA-bewerkingstechniek CRISPR-Cas
Oost, John van der; Groenen, Martien ; Maagd, Ruud de - \ 2017
Characterizing a thermostable Cas9 for bacterial genome editing and silencing
Mougiakos, Ioannis ; Mohanraju, Prarthana ; Bosma, Elleke F. ; Vrouwe, Valentijn ; Finger Bou, Max ; Naduthodi, Mihris I.S. ; Gussak, Alex ; Brinkman, Rudolf B.L. ; Kranenburg, Richard Van; Oost, John Van Der - \ 2017
Nature Communications 8 (2017)1. - ISSN 2041-1723
CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range.
Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9
Mougiakos, Ioannis ; Bosma, Elleke F. ; Weenink, Koen ; Vossen, Eric ; Goijvaerts, Kirsten ; Oost, John van der; Kranenburg, Richard van - \ 2017
ACS synthetic biology 6 (2017)5. - ISSN 2161-5063 - p. 849 - 861.
Bacillus smithii - bacteria - CRISPR/Cas9 - genome editing - homologous recombination - thermophiles

Well-developed genetic tools for thermophilic microorganisms are scarce, despite their industrial and scientific relevance. Whereas highly efficient CRISPR/Cas9-based genome editing is on the rise in prokaryotes, it has never been employed in a thermophile. Here, we apply Streptococcus pyogenes Cas9 (spCas9)-based genome editing to a moderate thermophile, i.e., Bacillus smithii, including a gene deletion, gene knockout via insertion of premature stop codons, and gene insertion. We show that spCas9 is inactive in vivo above 42 °C, and we employ the wide temperature growth range of B. smithii as an induction system for spCas9 expression. Homologous recombination with plasmid-borne editing templates is performed at 45-55 °C, when spCas9 is inactive. Subsequent transfer to 37 °C allows for counterselection through production of active spCas9, which introduces lethal double-stranded DNA breaks to the nonedited cells. The developed method takes 4 days with 90, 100, and 20% efficiencies for gene deletion, knockout, and insertion, respectively. The major advantage of our system is the limited requirement for genetic parts: only one plasmid, one selectable marker, and a promoter are needed, and the promoter does not need to be inducible or well-characterized. Hence, it can be easily applied for genome editing purposes in both mesophilic and thermophilic nonmodel organisms with a limited genetic toolbox and ability to grow at, or tolerate, temperatures of 37 and at or above 42 °C.

Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a
Swarts, Daan C. ; Oost, John van der; Jinek, Martin - \ 2017
Molecular Cell 66 (2017)2. - ISSN 1097-2765 - p. 221 - 233.e4.
Cas12a - Cas9 - Cpf1 - CRISPR RNA - CRISPR-Cas - crRNA processing - nuclease - R-loop - seed sequence - target DNA cleavage

The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies.

Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array
Zetsche, Bernd ; Heidenreich, Matthias ; Mohanraju, Prarthana ; Fedorova, Iana ; Kneppers, Jeroen ; Degennaro, Ellen M. ; Winblad, Nerges ; Choudhury, Sourav R. ; Abudayyeh, Omar O. ; Wu, Wen Y. ; Oost, John van der - \ 2017
Nature Biotechnology 35 (2017)1. - ISSN 1087-0156 - p. 31 - 34.

Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.

CRISPR-Cas9 gene editing : Delivery aspects and therapeutic potential
Oude Blenke, Erik ; Evers, Martijn J.W. ; Mastrobattista, Enrico ; Oost, John van der - \ 2016
Journal of Controlled Release 244 (2016). - ISSN 0168-3659 - p. 139 - 148.
CRISPR-Cas - CRISPR-Cas9 - Delivery systems - Ex vivo - Gene editing - Genome editing - In vivo - Therapeutic applications

The CRISPR-Cas9 gene editing system has taken the biomedical science field by storm, initiating rumors about future Nobel Prizes and heating up a fierce patent war, but also making significant scientific impact. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), together with CRISPR-associated proteins (Cas) are a part of the prokaryotic adaptive immune system and have successfully been repurposed for genome editing in mammalian cells. The CRISPR-Cas9 system has been used to correct genetic mutations and for replacing entire genes, opening up a world of possibilities for the treatment of genetic diseases. In addition, recently some new CRISPR-Cas systems have been discovered with interesting mechanistic variations. Despite these promising developments, many challenges have to be overcome before the system can be applied therapeutically in human patients and enabling delivery technology is one of the key challenges. Furthermore, the relatively high off-target effect of the system in its current form prevents it from being safely applied directly in the human body. In this review, the transformation of the CRISPR-Cas gene editing systems into a therapeutic modality will be discussed and the currently most realistic . in vivo applications will be highlighted.

Gummen in de genen
Oost, John van der; Schaart, Jan ; Smulders, Rene - \ 2016

De nieuwe technologie Crispr-Cas9 maakt het makkelijker dan ooit om erfelijk materiaal heel gericht te herschrijven. De ontwikkeling van deze gene editing gaat zo snel, dat wetgeving en ethiek moeite hebben het bij te benen. Is dit nu genmodifi catie of klassieke veredeling in de snelkookpan?

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems
Mohanraju, Prarthana ; Makarova, Kira S. ; Zetsche, Bernd ; Zhang, Feng ; Koonin, Eugene V. ; Oost, John van der - \ 2016
Science 353 (2016)6299. - ISSN 0036-8075 - 14 p.

Adaptive immunity had been long thought of as an exclusive feature of animals. However, the discovery of the CRISPR-Cas defense system, present in almost half of prokaryotic genomes, proves otherwise. Because of the everlasting parasite-host arms race, CRISPR-Cas has rapidly evolved through horizontal transfer of complete loci or individual modules, resulting in extreme structural and functional diversity. CRISPR-Cas systems are divided into two distinct classes that each consist of three types and multiple subtypes. We discuss recent advances in CRISPR-Cas research that reveal elaborate molecular mechanisms and provide for a plausible scenario of CRISPR-Cas evolution. We also briefly describe the latest developments of a wide range of CRISPR-based applications.

Next Generation Prokaryotic Engineering : The CRISPR-Cas Toolkit
Mougiakos, Ioannis ; Bosma, Elleke F. ; Vos, Willem M. de; Kranenburg, Richard van; Oost, John van der - \ 2016
Trends in Biotechnology 34 (2016)7. - ISSN 0167-7799 - p. 575 - 587.
Archaea - Bacteria - Cas9 - CRISPR-Cas - Genome editing - Recombineering

The increasing demand for environmentally friendly production processes of green chemicals and fuels has stimulated research in microbial metabolic engineering. CRISPR-Cas-based tools for genome editing and expression control have enabled fast, easy, and accurate strain development for established production platform organisms, such as Escherichia coli and Saccharomyces cerevisiae. However, the growing interest in alternative production hosts, for which genome editing options are generally limited, requires further developing such engineering tools. In this review, we discuss established and emerging CRISPR-Cas-based tools for genome editing and transcription control of model and non-model prokaryotes, and we analyse the possibilities for further improvement and expansion of these tools for next generation prokaryotic engineering. SpyCas9 has recently been established as an efficient counterselection system in combination with homologous recombination-based strategies for bacterial genome editing.Besides the traditionally used SpyCas9, other CRISPR-Cas systems (both heterologous and native) are currently being evaluated in bacteria for their editing potential.Catalytically inactive variants of CRISPR-Cas systems are used for transcriptional control in bacteria with great potential for fundamental research and applications.

Effects of Argonaute on gene expression in Thermus thermophilus
Swarts, D.C. ; Koehorst, J.J. ; Westra, E.R. ; Schaap, P.J. ; Oost, J. van der - \ 2015
PRJEB8709 - ERP009732 - Thermus thermophilus
To investigate if TtAgo also has the potential to control RNA levels, we analyzed RNA-seq data derived from cultures of four T. thermophilus strain HB27 variants: wild type, TtAgo knockout (Δago), and either strain transformed with a plasmid. Additionally we determined the effect of TtAgo on expression of plasmid-encoded RNA and plasmid DNA levels. Background Eukaryotic Argonaute proteins mediate RNA-guided RNA interference, allowing both regulation of host gene expression and defense against invading mobile genetic elements. Recently, it has become evident that prokaryotic Argonaute homologs mediate DNA-guided DNA interference, and play a role in host defense. Argonaute of the bacterium Thermus thermophilus (TtAgo) targets invading plasmid DNA during and after transformation. Using small interfering DNA guides, TtAgo can cleave single and double stranded DNAs. Although TtAgo additionally has been demonstrated to cleave RNA targets complementary to its DNA guide in vitro, RNA targeting by TtAgo has not been demonstrated in vivo. Methods To investigate if TtAgo also has the potential to control RNA levels, we analyzed RNA-seq data derived from cultures of four T. thermophilus strain HB27 variants: wild type, TtAgo knockout (Δago), and either strain transformed with a plasmid. Additionally we determined the effect of TtAgo on expression of plasmid-encoded RNA and plasmid DNA levels. Results In the absence of exogenous DNA (plasmid), TtAgo presence or absence had no effect on gene expression levels. When plasmid DNA is present, TtAgo reduces plasmid DNA levels 4-fold, and a corresponding reduction of plasmid gene transcript levels was observed. We therefore conclude that TtAgo interferes with plasmid DNA, but not with plasmid RNA. Interestingly, TtAgo presence stimulates expression of specific endogenous genes, but only when exogenous plasmid DNA was present. Specifically, the presence of TtAgo directly or indirectly stimulates expression of CRISPR loci and associated genes, some of which are involved in CRISPR adaptation. This suggests that TtAgo-mediated interference with plasmid DNA stimulates CRISPR adaptation.
Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System
Zetsche, Bernd ; Gootenberg, Jonathan S. ; Abudayyeh, Omar O. ; Slaymaker, Ian M. ; Makarova, Kira S. ; Essletzbichler, Patrick ; Volz, Sara E. ; Joung, Julia ; Oost, John van der; Regev, Aviv ; Koonin, Eugene V. ; Zhang, Feng - \ 2015
Cell 163 (2015)3. - ISSN 0092-8674 - p. 759 - 771.
The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Here, we report characterization of Cpf1, a putative class 2 CRISPR effector. We demonstrate that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, we identified two candidate enzymes from Acidaminococcus and Lachnospiraceae, with efficient genome-editing activity in human cells. Identifying this mechanism of interference broadens our understanding of CRISPR-Cas systems and advances their genome editing applications.
Analysis of protein-RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry
Sharma, Kundan ; Hrle, Ajla ; Kramer, Katharina ; Sachsenberg, Timo ; Staals, Raymond H.J. ; Randau, Lennart ; Marchfelder, Anita ; Oost, John van der; Kohlbacher, Oliver ; Conti, Elena ; Urlaub, Henning - \ 2015
Methods : a companion to Methods in enzymology 89 (2015). - ISSN 1046-2023 - p. 138 - 148.
Cas7 - CRISPR-Cas - Mass spectrometry - Protein-RNA interactions - UV cross-linking

Ribonucleoprotein (RNP) complexes play important roles in the cell by mediating basic cellular processes, including gene expression and its regulation. Understanding the molecular details of these processes requires the identification and characterization of protein-RNA interactions. Over the years various approaches have been used to investigate these interactions, including computational analyses to look for RNA binding domains, gel-shift mobility assays on recombinant and mutant proteins as well as co-crystallization and NMR studies for structure elucidation. Here we report a more specialized and direct approach using UV-induced cross-linking coupled with mass spectrometry. This approach permits the identification of cross-linked peptides and RNA moieties and can also pin-point exact RNA contact sites within the protein. The power of this method is illustrated by the application to different single- and multi-subunit RNP complexes belonging to the prokaryotic adaptive immune system, CRISPR-Cas (CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR associated). In particular, we identified the RNA-binding sites within three Cas7 protein homologs and mapped the cross-linking results to reveal structurally conserved Cas7 - RNA binding interfaces. These results demonstrate the strong potential of UV-induced cross-linking coupled with mass spectrometry analysis to identify RNA interaction sites on the RNA binding proteins.

An updated evolutionary classification of CRISPR-Cas systems
Makarova, K.S. ; Wolf, Y.I. ; Alkhnbashi, O.S. ; Brouns, S.J.J. ; Oost, John Van Der - \ 2015
Nature Reviews Microbiology 13 (2015)11. - ISSN 1740-1526 - p. 722 - 736.

The evolution of CRISPR-cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR-cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR-Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized.

CRISPR sabotage
Oost, John van der; Brouns, S.J.J. - \ 2015
Genome Biology 16 (2015)1. - ISSN 1474-7596

The biological arms race generally involves the rapid co-evolution of anti-virus systems in host organisms and of anti-anti-virus systems in their viral parasites. The CRISPR-Cas system is an example of a prokaryotic immune system in which such co-evolution occurs, as was recently demonstrated by the characterization of a set of viral anti-CRISPR proteins.

Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity
Charpentier, Emmanuelle ; Richter, Hagen ; Oost, John van der; White, Malcolm F. - \ 2015
FEMS Microbiology Reviews 39 (2015)3. - ISSN 0168-6445 - p. 428 - 441.
Cas5d - Cas6 - Cas9 - crRNA biogenesis - RNase III - TracrRNA

CRISPR-Cas is an RNA-mediated adaptive immune system that defends bacteria and archaea against mobile genetic elements. Short mature CRISPR RNAs (crRNAs) are key elements in the interference step of the immune pathway. A CRISPR array composed of a series of repeats interspaced by spacer sequences acquired from invading mobile genomes is transcribed as a precursor crRNA (pre-crRNA) molecule. This pre-crRNA undergoes one or two maturation steps to generate the mature crRNAs that guide CRISPR-associated (Cas) protein(s) to cognate invading genomes for their destruction. Different types of CRISPR-Cas systems have evolved distinct crRNA biogenesis pathways that implicate highly sophisticated processing mechanisms. In Types I and III CRISPR-Cas systems, a specific endoribonuclease of the Cas6 family, either standalone or in a complex with other Cas proteins, cleaves the pre-crRNA within the repeat regions. In Type II systems, the trans-acting small RNA (tracrRNA) base pairs with each repeat of the pre-crRNA to form a dual-RNA that is cleaved by the housekeeping RNase III in the presence of the protein Cas9. In this review, we present a detailed comparative analysis of pre-crRNA recognition and cleavage mechanisms involved in the biogenesis of guide crRNAs in the three CRISPR-Cas types.

Effects of Argonaute on Gene Expression in Thermus thermophilus
Swarts, D.C. ; Koehorst, J.J. ; Westra, E.R. ; Schaap, P.J. ; Oost, J. van der - \ 2015
PLoS ONE 10 (2015)4. - ISSN 1932-6203 - 13 p.
BACKGROUND: Eukaryotic Argonaute proteins mediate RNA-guided RNA interference, allowing both regulation of host gene expression and defense against invading mobile genetic elements. Recently, it has become evident that prokaryotic Argonaute homologs mediate DNA-guided DNA interference, and play a role in host defense. Argonaute of the bacterium Thermus thermophilus (TtAgo) targets invading plasmid DNA during and after transformation. Using small interfering DNA guides, TtAgo can cleave single and double stranded DNAs. Although TtAgo additionally has been demonstrated to cleave RNA targets complementary to its DNA guide in vitro, RNA targeting by TtAgo has not been demonstrated in vivo. METHODS: To investigate if TtAgo also has the potential to control RNA levels, we analyzed RNA-seq data derived from cultures of four T. thermophilus strain HB27 variants: wild type, TtAgo knockout (¿ago), and either strain transformed with a plasmid. Additionally we determined the effect of TtAgo on expression of plasmid-encoded RNA and plasmid DNA levels. RESULTS: In the absence of exogenous DNA (plasmid), TtAgo presence or absence had no effect on gene expression levels. When plasmid DNA is present, TtAgo reduces plasmid DNA levels 4-fold, and a corresponding reduction of plasmid gene transcript levels was observed. We therefore conclude that TtAgo interferes with plasmid DNA, but not with plasmid-encoded RNA. Interestingly, TtAgo presence stimulates expression of specific endogenous genes, but only when exogenous plasmid DNA was present. Specifically, the presence of TtAgo directly or indirectly stimulates expression of CRISPR loci and associated genes, some of which are involved in CRISPR adaptation. This suggests that TtAgo-mediated interference with plasmid DNA stimulates CRISPR adaptation
Structural biology. Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning
Taylor, D.W. ; Zhu, Y. ; Staals, R.H.J. ; Kornfeld, J.E. ; Shinkai, A. ; Oost, J. van der; Nogales, E. ; Doudna, J.A. - \ 2015
Science 348 (2015)6234. - ISSN 0036-8075 - p. 581 - 585.
Adaptive immunity in bacteria involves RNA-guided surveillance complexes that use CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target invasive nucleic acids for degradation. Whereas type I and type II CRISPR-Cas surveillance complexes target double-stranded DNA, type III complexes target single-stranded RNA. Near-atomic resolution cryo-electron microscopy reconstructions of native type III Cmr (CRISPR RAMP module) complexes in the absence and presence of target RNA reveal a helical protein arrangement that positions the crRNA for substrate binding. Thumblike ß hairpins intercalate between segments of duplexed crRNA:target RNA to facilitate cleavage of the target at 6-nucleotide intervals. The Cmr complex is architecturally similar to the type I CRISPR-Cascade complex, suggesting divergent evolution of these immune systems from a common ancestor.
Bacteriophage exclusion, a new defense system
Barrangou, R. ; Oost, J. van der - \ 2015
The EMBO Journal 34 (2015)2. - ISSN 0261-4189 - p. 134 - 135.
The ability to withstand viral predation is critical for survival of most microbes. Accordingly, a plethora of phage resistance systems has been identified in bacterial genomes (Labrie et al, 2010), including restriction-modification systems (R-M) (Tock & Dryden, 2005), abortive infection (Abi) (Chopin et al, 2005), Argonaute-based interference (Swarts et al, 2014), as well as clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein (Cas) adaptive immune system (CRISPR-Cas) (Barrangou & Marraffini, 2014; Van der Oost et al, 2014). Predictably, the dark matter of bacterial genomes contains a wealth of genetic gold. A study published in this issue of The EMBO Journal by Goldfarb et al (2015) unveils bacteriophage exclusion (BREX) as a novel, widespread bacteriophage resistance system that provides innate immunity against virulent and temperate phage in bacteria.
RNA-targeting by the Type III-A CRISPR-Cas complex of Thermus thermophilus
Staals, R.H.J. ; Zhu, Y. ; Taylor, D.W. ; Kornfeld, J.E. ; Sharma, K. ; Barendregt, A. ; Koehorst, J.J. ; Vlot, M. ; Neupane, N. ; Varossieau, K. ; Sakamoto, K. ; Suzuki, T. ; Schaap, P.J. ; Urlaub, H. ; Heck, A.J.R. ; Nogales, E. ; Doudna, J.A. ; Shinkai, A. ; Oost, J. van der - \ 2014
PRJEB7461 - ERP007191
crRNAs from the Thermus thermophilus CRISPR-Cas Csm complex
RNA Targeting by the Type III-A CRISPR-Cas Csm Complex of Thermus thermophilus
Staals, R.H.J. ; Zhu, Y. ; Taylor, D.W. ; Kornfeld, J.E. ; Sharma, K. ; Barendregt, A. ; Koehorst, J.J. ; Vlot, M. ; Neupane, N. ; Varossieau, K. ; Sakamoto, K. ; Suzuki, T. ; Schaap, P.J. ; Urlaub, H. ; Heck, A.J.R. ; Nogales, E. ; Doudna, J.A. ; Shinkai, A. ; Oost, J. van der - \ 2014
Molecular Cell 56 (2014)4. - ISSN 1097-2765 - p. 518 - 530.
guided surveillance complex - bacterial immune-system - adaptive immunity - mass-spectrometry - crystal-structure - escherichia-coli - haloferax-volcanii - antiviral defense - seed sequence - protein
CRISPR-Cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. Here we report the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). TtCsm is composed of five different protein subunits (Csm1–Csm5) with an uneven stoichiometry and a single crRNA of variable size (35–53 nt). The TtCsm crRNA content is similar to the Type III-B Cmr complex, indicating that crRNAs are shared among different subtypes. A negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes. crRNA-protein crosslinking studies show extensive contacts between the Csm3 backbone and the bound crRNA. We show that, like TtCmr, TtCsm cleaves complementary target RNAs at multiple sites. Unlike Type I complexes, interference by TtCsm does not proceed via initial base pairing by a seed sequence.
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