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10.1038/nature15544 http://scihub22266oqcxt.onion/10.1038/nature15544 C4859810!4859810!26524520     free     free     free Warning: file_get_contents(https://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=26524520&cmd=llinks): Failed to open stream: HTTP request failed! HTTP/1.1 429 Too Many Requests in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 215 Deprecated: Implicit conversion from float 209.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534       Nature 2015 ; 527 (7576): 110-3 Nephropedia Template TP {{tp|p=26524520|t=2015. Conformational control of DNA target cleavage by CRISPR?Cas9 |pdf=|usr=}}{{26524520}} gab.com Text Conformational control of DNA target cleavage by CRISPR Cas9 JO: Nature http://www.kidney.de/pget.php?&tw=1&pmid=26524520 #FOAvip Cas9 is an RNA-guided DNA endonuclease that targets foreign DNA for destruction as part of a bacterial adaptive immune system mediated by CRISPR (clustered regularly interspaced short palindromic repeats)1,2. Together with single-guide RNAs (sgRNA)3, Cas9 also functions as a powerful genome engineering tool in plants and animals4?6, and efforts are underway to increase the efficiency and specificity of DNA targeting for potential therapeutic applications7,8. Studies of off-target effects have shown that DNA binding is far more promiscuous than DNA cleavage9?11, yet the molecular cues that govern strand scission have not been elucidated. Here we show that the conformational state of the HNH nuclease domain directly controls DNA cleavage activity. Using intramolecular Förster resonance energy transfer (FRET) experiments to detect relative orientations of the Cas9 catalytic domains when associated with on- and off-target DNA, we find that DNA cleavage efficiencies scale with the extent to which the HNH domain samples an activated conformation. We furthermore uncover a surprising mode of allosteric communication that ensures concerted firing of both Cas9 nuclease domains. Our results highlight a proofreading mechanism beyond initial PAM recognition12 and RNA?DNA base-pairing3 that serves as a final specificity checkpoint before DNA double-strand break formation. Twit Text FOAVip Conformational control of DNA target cleavage by CRISPR Cas9 ????Nature http://www.kidney.de/pget.php?&tw=1&pmid=26524520 #FOAvip Twit Text # Free Paper on # # # # # LINK http://www.kidney.de/pget.php?&tw=1&pmid=26524520 #FOAvip English Wikipedia <ref>{{Cite pmid|26524520}}</ref> Conformational control of DNA target cleavage by CRISPR?Cas9 #MMPMID26524520 Sternberg SH; LaFrance B; Kaplan M; Doudna JA Nature 2015[Nov]; 527 (7576): 110-3 PMID26524520show ga Cas9 is an RNA-guided DNA endonuclease that targets foreign DNA for destruction as part of a bacterial adaptive immune system mediated by CRISPR (clustered regularly interspaced short palindromic repeats)1,2. Together with single-guide RNAs (sgRNA)3, Cas9 also functions as a powerful genome engineering tool in plants and animals4?6, and efforts are underway to increase the efficiency and specificity of DNA targeting for potential therapeutic applications7,8. Studies of off-target effects have shown that DNA binding is far more promiscuous than DNA cleavage9?11, yet the molecular cues that govern strand scission have not been elucidated. Here we show that the conformational state of the HNH nuclease domain directly controls DNA cleavage activity. Using intramolecular Förster resonance energy transfer (FRET) experiments to detect relative orientations of the Cas9 catalytic domains when associated with on- and off-target DNA, we find that DNA cleavage efficiencies scale with the extent to which the HNH domain samples an activated conformation. We furthermore uncover a surprising mode of allosteric communication that ensures concerted firing of both Cas9 nuclease domains. Our results highlight a proofreading mechanism beyond initial PAM recognition12 and RNA?DNA base-pairing3 that serves as a final specificity checkpoint before DNA double-strand break formation. äConformational control of DNA target cleavage by CRISPR?Cas9 (epmc).pdf DeepDyve Pubget Overpricing