RNA-activated DNA cleavage by the Type III-B CRISPR–Cas effector complex

Estrella, Michael; Kuo, Fang Ting; Bailey, Scott

doi:10.1101/gad.273722.115

Cited by 182 publications

(238 citation statements)

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“…A single protein in Type III-A systems, Csm5, or two proteins in Type III-B systems, Cmr1 and Cmr6, cap the backbone filament at the head of the complex [5] (Fig 1A). The Cas10 protein typically contains an HD nuclease domain and palm polymerase domain, and is thought to be the subunit responsible for DNA cleavage activity [1,6–11]. The overall architecture of Type III complexes is similar to that of the more well-studied Type I “Cascade” complex, but unlike Cascade, which recruits a trans-acting Cas3 nuclease/helicase to degrade double-stranded DNA (dsDNA), the catalytic components are constitutively present in Type III complexes [6,10–16].…”

Section: Introductionmentioning

confidence: 99%

RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System

2017

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CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA) to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus) Type III-A Csm complex (TthCsm) with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA) by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (5′ tag) of the crRNA and the 3′ flanking region of the ssRNA. Mutation of the histidine-aspartate (HD) nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csm complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA) during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryo-electron microscopy and x-ray crystallography.

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Section: Introductionmentioning

confidence: 99%

RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System

2017

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“…However, it has recently been demonstrated that both Type III complexes are transcription-dependent DNA nucleases [83,103], i.e. they initially recognize their target through specific interaction of the crRNA guide with a complementary nascent mRNA, after which cleavage occurs of the flanking DNA sequences [104][105][106][107][108][109]. Robust interference by these systems relies on the concerted cleavage of the transcript RNA and the transcribed DNA.…”

Section: Target Interferencementioning

confidence: 99%

“…The Cas7-like backbone subunits (Csm3, Cmr4) are responsible for the ribonuclease activity, typically resulting in cleavage of the target RNA at 6 nucleotide intervals [83,98,102,103,[110][111][112]. Binding of the Cmr complex to its complementary RNA target induces a conformational change [35,98] that results in activation of the Cas10 DNAcleaving subunit (Csm1/Cmr2) [105,106,108]. Disruption of the ribonucleases active sites (in Csm3/Cmr4), at least in some cases, does not hamper the activation of the DNA nuclease activity of the complexes [103,105].…”

Section: Target Interferencementioning

confidence: 99%

“…Binding of the Cmr complex to its complementary RNA target induces a conformational change [35,98] that results in activation of the Cas10 DNAcleaving subunit (Csm1/Cmr2) [105,106,108]. Disruption of the ribonucleases active sites (in Csm3/Cmr4), at least in some cases, does not hamper the activation of the DNA nuclease activity of the complexes [103,105]. Exonucleolytic cleavage of single-stranded DNA and RNA by recombinant S. epidermidis Csm1 (Cas10), T. maritima and P. furiosus Cmr2 has been demonstrated in vitro [105,106,113].…”

Section: Target Interferencementioning

confidence: 99%

“…Disruption of the ribonucleases active sites (in Csm3/Cmr4), at least in some cases, does not hamper the activation of the DNA nuclease activity of the complexes [103,105]. Exonucleolytic cleavage of single-stranded DNA and RNA by recombinant S. epidermidis Csm1 (Cas10), T. maritima and P. furiosus Cmr2 has been demonstrated in vitro [105,106,113]. In the S. epidermidis system, a Csx1 ortholog (Csm6) provides an auxiliary RNA-targeting activity that operates in conjunction with the RNA-and DNA-targeting endonuclease activities of the Csm effector complex [114][115][116]; in the P. furiosus Cmr system Csx1 appears not to be an essential component [103].…”

Section: Target Interferencementioning

confidence: 99%

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Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

Mohanraju

Makarova

Zetsche

et al. 2016

Science

555

460

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Background: Prokaryotes have evolved multiple systems to combat invaders such as viruses and plasmids. Examples of such defence systems include receptor masking, restriction-modification (R-M systems), DNA interference (Argonaute), bacteriophage exclusion (BREX or PGL) and abortive infection, all of which act in an innate, non-specific manner. In addition, prokaryotes have evolved adaptive, heritable immune systems, i.e. clustered regularly interspaced palindromic repeats (CRISPR) and the CRISPR-associated proteins (CRISPR-Cas). Adaptive immunity is conferred by the integration of DNA sequences from an invading element into the CRISPR array (adaptation), which is transcribed into long pre-CRISPR (pre-cr) RNAs and processed into short crRNAs (expression), which guide Cas proteins to specifically degrade the cognate DNA on subsequent exposures (interference). Advances:A plethora of distinct CRISPR-Cas systems are represented in genomes of most archaea and almost half of the bacteria. The latest CRISPR-Cas classification scheme delineates two classes that are each subdivided into three types. Integration of biochemistry and molecular genetics has contributed significantly to revealing many of the unique features of the variant CRISPR-Cas types. Additionally, structural analysis and single molecule studies have further advanced our understanding of the molecular basis of CRISPR-Cas functionality. Recent progress includes relevant steps in the adaptation stage, when fragments of foreign DNA are processed and incorporated as new spacers into the CRISPR array. In addition, three novel CRISPR-Cas types (IV, V, and VI) have been identified, and in particular, the type V interference complexes have been experimentally characterized. Moreover, the ability to easily program sequence-specific DNA targeting and cleavage by CRISPRCas components, as demonstrated for Cas9 and Cpf1, allows for the application of CRISPR-Cas components as highly effective tools for genetic engineering and gene regulation in a wide range of eukaryotes and prokaryotes.The pressing issue of off-target cleavage by the Cas9 nuclease is being actively addressed using structure-guided engineering.Outlook: Although our understanding of the CRISPR-Cas system has increased tremendously over the past few years, much remains to be done. About the evolution of CRISPR-Cas systems, the continuing discovery of novel CRISPR-Cas variants will provide direct tests of the recently proposed modular scenario. The recent discovery and characterization of new CRISPR-Cas types with many unique features implies that our current knowledge has relatively limited power for predicting the functional details of distantly related variants. Hence, newly discovered CRISPR-Cas systems need to be thoroughly dissected with the aforementioned multi-disciplinary approaches to gain insight in their biological role, to unravel their molecular mechanism, and to harness their potential for biotechnology. As to the biology, key outstanding questions include the ecological roles of microbial ...

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CRISPR Cas

Šviković

2022

Genome Editing in Drug Discovery

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RNA-activated DNA cleavage by the Type III-B CRISPR–Cas effector complex

Cited by 182 publications

References 47 publications

RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System

RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

CRISPR Cas

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