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 <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.
+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. These structural observations explain the RNA-guided DNA targeting mechanism of SaCas9. The C-terminal region of the REC lobe interacts with the PAM-distal region of the heteroduplex, whereas the N-terminal region of the REC lobe interacts with the repeat:anti-repeat duplex and the PAM-proximal region of the heteroduplex. Notably, the C-terminal region of the REC lobe of SaCas9 shares structural similarity with those of SpCas9 (PDB: [[4un3]], 26% identity, rmsd of 1.9 A˚ for 177 equivalent Ca atoms) and AnCas9 (PDB: [[4oge]], 16% identity, rmsd of 3.2 A˚ for 167 equivalent Ca atoms). These structural findings suggested that the Cas9 orthologs recognize the PAM-distal region of the guide:target heteroduplex in a similar manner.
 =See aslo=
 *[[Cas9]]