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10.1038/ncomms12778 http://scihub22266oqcxt.onion/10.1038/ncomms12778 C5027287!5027287!27624851     free     free     free Deprecated: Implicit conversion from float 219.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 219.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 219.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 219.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 219.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534       Nat+Commun 2016 ; 7 (ä): ä Nephropedia Template TP {{tp|p=27624851|t=2016. Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9 |pdf=|usr=}}{{27624851}} gab.com Text Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9 JO: Nat Commun http://www.kidney.de/pget.php?&tw=1&pmid=27624851 #FOAvip Binding specificity of Cas9?guide RNA complexes to DNA is important for genome-engineering applications; however, how mismatches influence target recognition/rejection kinetics is not well understood. Here we used single-molecule FRET to probe real-time interactions between Cas9?RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence; however, the dissociation rate greatly increases from <0.006?s?1 to >2?s?1 upon introduction of mismatches proximal to protospacer-adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 11 base pairs in length, which prevent DNA cleavage, still allow formation of a stable complex (dissociation rate <0.006?s?1), suggesting that extremely slow rejection could sequester Cas9?RNA, increasing the Cas9 expression level necessary for genome-editing, thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading. Twit Text FOAVip Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9 ????Nat Commun http://www.kidney.de/pget.php?&tw=1&pmid=27624851 #FOAvip Twit Text # Free Paper on # # # # # LINK http://www.kidney.de/pget.php?&tw=1&pmid=27624851 #FOAvip English Wikipedia <ref>{{Cite pmid|27624851}}</ref> Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9 #MMPMID27624851 Singh D; Sternberg SH; Fei J; Doudna JA; Ha T Nat Commun 2016[]; 7 (ä): ä PMID27624851show ga Binding specificity of Cas9?guide RNA complexes to DNA is important for genome-engineering applications; however, how mismatches influence target recognition/rejection kinetics is not well understood. Here we used single-molecule FRET to probe real-time interactions between Cas9?RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence; however, the dissociation rate greatly increases from <0.006?s?1 to >2?s?1 upon introduction of mismatches proximal to protospacer-adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 11 base pairs in length, which prevent DNA cleavage, still allow formation of a stable complex (dissociation rate <0.006?s?1), suggesting that extremely slow rejection could sequester Cas9?RNA, increasing the Cas9 expression level necessary for genome-editing, thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading. äReal-time observation of DNA recognition and rejection by the RNA-guid(epmc).pdf DeepDyve Pubget Overpricing