Titlebook: CRISPR; Methods and Protocol Magnus Lundgren,Emmanuelle Charpentier,Peter C. Fi Book 2015 Springer Science+Business Media New York 2015 CRI

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In Vitro Co-reconstitution of Cas Protein Complexes,n of the mechanistic details and the protein interaction partners requires production of recombinant Cas proteins. However, these proteins are often produced as inactive inclusion bodies. Here, we present a detailed protocol for the isolation and purification of insoluble Cas proteins. Guidelines fo

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Analysis of CRISPR Pre-crRNA Cleavage,Cas system by incubation of radiolabeled model RNAs with recombinant CRISPR-associated (Cas) endoribonucleases, followed by denaturing polyacrylamide gel electrophoresis (PAGE) of the products. Determination of cleavage position is based on comparison with RNase T1 digestion and base hydrolysis prod

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Computational Detection of CRISPR/crRNA Targets,ys specifically determines the targets in invader genomes. These spacers provide the short specific RNA nucleotide sequences within the guide crRNAs. In addition to complementarity in the spacer–target (protospacer) interaction, short flanking protospacer adjacent motifs (PAMs), or mismatching flank

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High-Throughput CRISPR Typing of , Complex and , Serotype Typhimurium,inical isolates at a phylogeographic level. For other pathogens, such as ., recent studies suggest that specifically designed spoligotyping techniques could be interesting for public health purposes. Spoligotyping was in its original format a reverse line-blot hybridization method using capture prob

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Spacer-Based Macroarrays for CRISPR Genotyping, loci in bacteria. To date, it was used almost exclusively for . and was named spoligotyping (spacer oligonucleotides typing). Here, we describe the pipeline of this approach that includes search of loci and selection of spacers, preparation of the membrane with immobilized probes and spoligotyping

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Analysis of crRNA Using Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC ESI MS)id chromatography interfaced with electrospray ionization mass spectrometry (LC ESI MS). The direct purification of crRNA from the Cascade-crRNA complex was performed using denaturing ion pair reverse phase chromatography. Following purification of the crRNA, the intact mass was determined by LC ESI

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