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Crystallization and preliminary X-ray diffraction analysis of the CRISPR-Cas RNA-silencing Cmr complex.

Crystallization and preliminary X-ray diffraction analysis of the CRISPR-Cas RNA-silencing Cmr complex.

Acta Crystallogr F Struct Biol Commun. 2015 Jun 1;71(Pt 6):735-740

Authors: Osawa T, Inanaga H, Numata T

Abstract
Clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNA (crRNA) and CRISPR-associated (Cas) proteins constitute a prokaryotic adaptive immune system (CRISPR-Cas system) that targets and degrades invading genetic elements. The type III-B CRISPR-Cas Cmr complex, composed of the six Cas proteins (Cmr1-Cmr6) and a crRNA, captures and cleaves RNA complementary to the crRNA guide sequence. Here, a Cmr1-deficient functional Cmr (CmrΔ1) complex composed of Pyrococcus furiosus Cmr2-Cmr3, Archaeoglobus fulgidus Cmr4-Cmr5-Cmr6 and the 39-mer P. furiosus 7.01-crRNA was prepared. The CmrΔ1 complex was cocrystallized with single-stranded DNA (ssDNA) complementary to the crRNA guide by the vapour-diffusion method. The crystals diffracted to 2.1 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the triclinic space group P1, with unit-cell parameters a = 75.5, b = 76.2, c = 139.2 Å, α = 90.3, β = 104.8, γ = 118.6°. The asymmetric unit of the crystals is expected to contain one CmrΔ1-ssDNA complex, with a Matthews coefficient of 2.03 Å(3) Da(-1) and a solvent content of 39.5%.

PMID: 26057804 [PubMed – as supplied by publisher]

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Crystal structure of the RNA-guided immune surveillance Cascade complex in Escherichia coli.

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Crystal structure of the RNA-guided immune surveillance Cascade complex in Escherichia coli.

Nature. 2014 Nov 6;515(7525):147-50

Authors: Zhao H, Sheng G, Wang J, Wang M, Bunkoczi G, Gong W, Wei Z, Wang Y

Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated (Cas) proteins form the CRISPR/Cas system to defend against foreign nucleic acids of bacterial and archaeal origin. In the I-E subtype CRISPR/Cas system, eleven subunits from five Cas proteins (CasA1B2C6D1E1) assemble along a CRISPR RNA (crRNA) to form the Cascade complex. Here we report on the 3.05 Å crystal structure of the 405-kilodalton Escherichia coli Cascade complex that provides molecular details beyond those available from earlier lower-resolution cryo-electron microscopy structures. The bound 61-nucleotide crRNA spans the entire 11-protein subunit-containing complex, where it interacts with all six CasC subunits (named CasC1-6), with its 5′ and 3′ terminal repeats anchored by CasD and CasE, respectively. The crRNA spacer region is positioned along a continuous groove on the concave surface generated by the aligned CasC1-6 subunits. The five long β-hairpins that project from individual CasC2-6 subunits extend across the crRNA, with each β-hairpin inserting into the gap between the last stacked base and its adjacent splayed counterpart, and positioned within the groove of the preceding CasC subunit. Therefore, instead of continuously stacking, the crRNA spacer region is divided into five equal fragments, with each fragment containing five stacked bases flanked by one flipped-out base. Each of those crRNA spacer fragments interacts with CasC in a similar fashion. Furthermore, our structure explains why the seed sequence, with its outward-directed bases, has a critical role in target DNA recognition. In conclusion, our structure of the Cascade complex provides novel molecular details of protein-protein and protein-RNA alignments and interactions required for generation of a complex mediating RNA-guided immune surveillance.

PMID: 25118175 [PubMed – indexed for MEDLINE]

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RCas9: A Programmable RNA Editing Tool

newscenter.lbl.gov
– lcyarris
A powerful scientific tool for editing the DNA instructions in a genome can now also be applied to RNA, the molecule that translates DNA’s genetic instructions into the production of proteins. A team of researchers with Berkeley Lab and the University of California (UC) Berkeley has demonstrated a means by which the CRISPR/Cas9 protein complex can be programmed to recognize and cleave RNA at sequence-specific target sites. This finding has the potential to transform the study of RNA function by…  show all text
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@idtdna: Boost for Synthetic #Biology: Powerful #DNA editing tool can now be programmed to edit RNA. bit.ly/cas9r http://ift.tt/1tnrJ9j
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Modified CRISPR Expands Targets | The Scientist Magazine®

Modified CRISPR Expands Targets | The Scientist Magazine®.

Modified CRISPR Expands Targets | The Scientist Magazine®

Indyplus video: Crispr technique – Science – News – The Independent

Indyplus video: Crispr technique – Science – News – The Independent.

Exclusive: Jaw-dropping breakthrough hailed as landmark in fight against hereditary diseases as CRISPR technique heralds genetic revolution – Science – News – The Independent

Exclusive: Jaw-dropping breakthrough hailed as landmark in fight against hereditary diseases as Crispr technique heralds genetic revolution – Science – News – The Independent.

Indyplus video: Crispr technique – Science – News – The Independent.

CRISPR Genome Engineering Resources | learn, share, and discuss

CRISPR Genome Engineering Resources | learn, share, and discuss.

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