CRISPR-Cas9: Difference between revisions

The <scene name='74/742625/Cv6/32'>repeat:anti-repeat duplex is recognized by the REC and WED domains, primarily through interactions between the protein and the sugar-phosphate backbone</scene>. Consistent with our data showing that the distorted repeat:anti-repeat duplex is critical for Cas9-catalyzed DNA cleavage, the <scene name='74/742625/Cv6/33'>internal loop is recognized by the WED domain</scene>. The 2'-OH of C30 hydrogen bonds with <scene name='74/742625/Cv6/34'>Tyr868</scene>, and the backbone phosphate groups of U31, C45, and U46 interact with <scene name='74/742625/Cv6/35'>Lys870, Arg792, and Lys881</scene>, respectively. These structural observations explain the structure-dependent recognition of the repeat:anti-repeat duplex by SaCas9. Stem loop 1 is recognized by the bridge helix and the REC lobe. The phosphate backbone of <scene name='74/742625/Cv6/38'>stem loop 1</scene> interacts with the bridge helix (Arg47, Arg54, Arg55, Arg58, and Arg59) and the REC lobe (Arg209, Gly216, and Ser219). The 2'-OH of A63 hydrogen bonds with His62. The flipped-out U64 is recognized by Arg209 and Glu213 via stacking and hydrogen-bonding interactions, respectively. A55 is extensively recognized by the phosphate lock loop. The N6, N7, and 2'-OH of A55 hydrogen bond with Asn780/Arg781, Leu783, and Lys906, respectively. Lys57 interacts with the backbone phosphate group between C54 and A55, and the side chain of Leu783 forms hydrophobic contacts with the nucleobases of A55 and A56. The phosphate backbone of the linker region electrostatically interacts with the RuvC domain (Arg452, Lys459, and Arg774) and the phosphate lock loop (Arg781), and the nucleobase of G70 stacks with the side chain of Arg47 on the bridge helix.