CRISPR-Cas9: Difference between revisions

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Alexander Berchansky, Michal Harel

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 '''Recognition Mechanism of the Guide:Target Heteroduplex'''
-The <scene name='74/742625/Cv6/17'>guide:target heteroduplex is accommodated in the central channel</scene> formed between the REC and NUC lobes. The sugar-phosphate backbone of the PAM-distal region (A3–U6) of the sgRNA interacts with the <scene name='74/742625/Cv6/20'>REC lobe (Thr238, Tyr239, Lys248, Tyr256, Arg314, Asn394, and Gln414)</scene>. In SpCas9 and SaCas9, the RNA–DNA base pairing in the 8 bp PAM-proximal ‘‘seed’’ region in the guide:target heteroduplex is critical for Cas9-catalyzed DNA cleavage. Consistent with this, the phosphate backbone of the sgRNA seed region (C13–C20) is extensively recognized by the bridge helix (Asn44, Arg48, Arg51, Arg55, Arg59, and Arg60) and the REC lobe (Arg116, Gly117, Arg165, Gly166, Asn169, and Arg209) (Figures 3 and 4A), as in the case of SpCas9.
+The <scene name='74/742625/Cv6/17'>guide:target heteroduplex is accommodated in the central channel</scene> formed between the REC and NUC lobes. The sugar-phosphate backbone of the PAM-distal region (A3–U6) of the sgRNA interacts with the <scene name='74/742625/Cv6/20'>REC lobe (Thr238, Tyr239, Lys248, Tyr256, Arg314, Asn394, and Gln414)</scene>. In SpCas9 and SaCas9, the RNA–DNA base pairing in the 8 bp PAM-proximal ‘‘seed’’ region in the guide:target heteroduplex is critical for Cas9-catalyzed DNA cleavage. Consistent with this, the phosphate backbone of the sgRNA seed region (C13–C20) is extensively recognized <scene name='74/742625/Cv6/23'>by the bridge helix (Asn44, Arg48, Arg51, Arg55, Arg59, and Arg60) and the REC lobe (Arg116, Gly117, Arg165, Gly166, Asn169, Arg208, Arg209, and Tyr211)</scene>, as in the case of SpCas9.
 =See aslo=
 *[[Cas9]]