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10.1038/nbt.3528 http://scihub22266oqcxt.onion/10.1038/nbt.3528 C4900928!4900928!27136077     free     free     free Deprecated: Implicit conversion from float 229.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 229.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 229.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534       Nat+Biotechnol 2016 ; 34 (6): 646-51 Nephropedia Template TP {{tp|p=27136077|t=2016. Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch |pdf=|usr=}}{{27136077}} gab.com Text Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch JO: Nat Biotechnol http://www.kidney.de/pget.php?&tw=1&pmid=27136077 #FOAvip The CRISPR-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disrupting the enzyme?s binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the Estrogen Receptor ? Ligand Binding Domain. This protein displayed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9. Twit Text FOAVip Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch ????Nat Biotechnol http://www.kidney.de/pget.php?&tw=1&pmid=27136077 #FOAvip Twit Text # Free Paper on # # # # # LINK http://www.kidney.de/pget.php?&tw=1&pmid=27136077 #FOAvip English Wikipedia <ref>{{Cite pmid|27136077}}</ref> Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch #MMPMID27136077 Oakes BL; Nadler DC; Flamholz A; Fellmann C; Staahl BT; Doudna JA; Savage DF Nat Biotechnol 2016[Jun]; 34 (6): 646-51 PMID27136077show ga The CRISPR-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disrupting the enzyme?s binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the Estrogen Receptor ? Ligand Binding Domain. This protein displayed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9. äProfiling of engineering hotspots identifies an allosteric CRISPR-Cas9(epmc).pdf DeepDyve Pubget Overpricing