Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection

East-Seletsky, Alexandra; O’Connell, Mitchell R.; Knight, Spencer C.; Burstein, David; Cate, J.H.D.; Tjian, Robert; Doudna, Jennifer A.

doi:10.1038/nature19802

Cited by 880 publications

(995 citation statements)

References 35 publications

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“…In addition, it was found recently that C2c2 is a programmable RNA-guided RNA-targeting CRISPR effector (56). These findings suggest that the CRISPR/Cas system can be used to develop RNA-targeting tools (55)(56)(57). Compared with the CRISPR/Cas system that uses RNA-mediated base pairing to recognize RNA targets, SOT1 relies upon PPR-RNA interactions to recognize RNA targets.…”

Section: Discussionmentioning

confidence: 99%

PPR-SMR protein SOT1 has RNA endonuclease activity

Zhou

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Numerous attempts have been made to identify and engineer sequence-specific RNA endonucleases, as these would allow for efficient RNA manipulation. However, no natural RNA endonuclease that recognizes RNA in a sequence-specific manner has been described to date. Here, we report that SUPPRESSOR OF THYLAKOID FORMATION 1 (SOT1), an Arabidopsis pentatricopeptide repeat (PPR) protein with a small MutS-related (SMR) domain, has RNA endonuclease activity. We show that the SMR moiety of SOT1 performs the endonucleolytic maturation of 23S and 4.5S rRNA through the PPR domain, specifically recognizing a 13-nucleotide RNA sequence in the 5′ end of the chloroplast 23S-4.5S rRNA precursor. In addition, we successfully engineered the SOT1 protein with altered PPR motifs to recognize and cleave a predicted RNA substrate. Our findings point to SOT1 as an exciting tool for RNA manipulation.photosynthesis | PPR-SMR protein | RNA endonuclease | rRNA biogenesis S equence-specific RNA endonucleases are crucial to establishing RNA manipulation technology (1). Compared with DNA editing, RNA manipulation could be more useful and reversible because it does not result in permanent changes to the genome. In addition, sequence-specific RNA endonucleases could potentially be used as an RNA silencing tool to complement RNAi, because RNAi is sometimes ineffective in certain organisms and RNAi machinery is not present in cellular compartments such as chloroplasts and mitochondria. Despite extensive investigations, a natural RNA endonuclease that recognizes RNA in an intrinsic sequence-specific manner has not yet been identified.Pentatricopeptide repeat (PPR) proteins exist in eukaryotes, have greatly expanded in terrestrial plants, and take part in most RNA metabolic processes in organelles (2-5). The PPR domain can specifically recognize RNAs in an intrinsic sequence-specific manner (5-8). The 2nd, 5th, and 35th (or 1st, 4th, and 34th or 3rd, 6th, and 1st in other numbering systems) residues at each repeat are considered to be RNA selection "codes" (9-11). Based on these codes, several PPR proteins have been successfully modified to recognize predictable RNA targets (9,(12)(13)(14)(15)(16)(17).The small MutS-related (SMR) domain was originally identified at the C terminus of MutS2 in the cyanobacterium Synechocystis (18). SMR proteins are widely distributed in almost all organisms (19). Recent studies demonstrated the SMR domain exhibits DNA nicking nuclease activity in vitro (20-25). Furthermore, a C-terminal SMR domain in Leishmania donovani S-phase mRNA cycling sequence binding protein (CSBP) has RNA cleavage activity in vitro (26). These findings suggest that the SMR domain has nuclease activity.Interestingly, a small protein family containing both PPR and SMR domains was recently described (27). PPR-SMR proteins are found mainly in land plants. Arabidopsis thaliana contains eight PPR-SMR proteins localized to the organelles, including mitochondria and chloroplasts (27). Currently, four PPR-SMR proteins have been characterized. They play ...

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Section: Discussionmentioning

confidence: 99%

PPR-SMR protein SOT1 has RNA endonuclease activity

Zhou

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Remarkably, in the case of the type V-B effectors, both target and nontarget DNA strands are cleaved by the RuvC-like endonuclease domain, which undergoes a major conformational change triggered by the initial, nontarget strand cleavage (98, 182). Notably, the effector nucleases of type V-A and type VI are also responsible for the processing of pre-crRNA yielding mature crRNA guides; the catalytic domains and sites responsible for crRNA maturation remain poorly characterized but are clearly distinct from those involved in target cleavage (37, 44, 98). These findings are in sharp contrast to processing in type II, which involves RNase III, a ubiquitous bacterial enzyme (24), and emphasize the striking diversity of CRISPR-Cas molecular mechanisms.…”

Section: Adaptive Immunity: the Crispr-cas Systemmentioning

confidence: 99%

Evolutionary Genomics of Defense Systems in Archaea and Bacteria

Koonin

Makarova²,

Wolf³

2017

Annu. Rev. Microbiol.

268

235

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Evolution of bacteria and archaea involves an incessant arms race against an enormous diversity of genetic parasites. Accordingly, a substantial fraction of the genes in most bacteria and archaea are dedicated to antiparasite defense. The functions of these defense systems follow several distinct strategies, including innate immunity; adaptive immunity; and dormancy induction, or programmed cell death. Recent comparative genomic studies taking advantage of the expanding database of microbial genomes and metagenomes, combined with direct experiments, resulted in the discovery of several previously unknown defense systems, including innate immunity centered on Argonaute proteins, bacteriophage exclusion, and new types of CRISPR-Cas systems of adaptive immunity. Some general principles of function and evolution of defense systems are starting to crystallize, in particular, extensive gain and loss of defense genes during the evolution of prokaryotes; formation of genomic defense islands; evolutionary connections between mobile genetic elements and defense, whereby genes of mobile elements are repeatedly recruited for defense functions; the partially selfish and addictive behavior of the defense systems; and coupling between immunity and dormancy induction/programmed cell death.

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“…Type VI CRISPR-Cas systems include a single protein, Cas13 (formerly C2c2), that when assembled with a CRISPR-RNA (crRNA) forms a crRNA-guided RNA-targeting effector complex (Abudayyeh et al, 2016; East-Seletsky et al, 2016; Konermann et al, 2018; Shmakov et al, 2015; Smargon et al, 2017; Yan et al, 2018). Cas13 proteins are classified into distinct subfamilies (Cas13a-d), and all Cas13 proteins studied to date possess two enzymatically distinct RNase activities that are required for optimal interference (Abudayyeh et al, 2016; East-Seletsky et al, 2016; Konermann et al, 2018; Smargon et al, 2017; Yan et al, 2018). First, upon binding a precursor-crRNA (pre-crRNA), Cas13 cleaves within the crRNA direct repeat in a pre-crRNA array to form mature Cas13-crRNA complexes (East-Seletsky et al, 2016; East-Seletsky et al, 2017; Konermann et al, 2018; Smargon et al, 2017; Yan et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a

et al. 2018

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SUMMARY CRISPR-Cas13a enzymes are RNA-guided, RNA-activated ribonucleases. Their properties have been exploited as powerful tools for RNA detection, RNA imaging and RNA regulation. However, the relationship between target RNA binding and HEPN (higher-eukaryotes-and-prokaryotes nucleotide-binding)- domain nuclease activation is not well understood. Using sequencing experiments coupled with in vitro biochemistry, we find that Cas13a’s target RNA binding affinity and HEPN-nuclease activity are differentially affected by the number of and position of mismatches between the guide and target. We identify a central ‘binding seed’ where perfect base pairing is absolutely required for target binding, and a separate ‘nuclease switch’ where imperfect base-pairing results in tight binding but no HEPN-nuclease activation. These results demonstrate that the binding and cleavage activities of Cas13a are decoupled, highlighting a complex specificity landscape. Our findings underscore a need to consider the range of effects off-target recognition has on Cas13a’s RNA binding and cleavage behavior for RNA-targeting tool development.

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Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection

Cited by 880 publications

References 35 publications

PPR-SMR protein SOT1 has RNA endonuclease activity

PPR-SMR protein SOT1 has RNA endonuclease activity

Evolutionary Genomics of Defense Systems in Archaea and Bacteria

RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a

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