CRISPR-Cas9: Difference between revisions
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'''Recognition Mechanism of the sgRNA Scaffold''' | '''Recognition Mechanism of the sgRNA Scaffold''' | ||
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. | 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 stem loop 1 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 (Figure 4D). | |||
The N6, N7, and 20 | |||
-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 (Figure 4D). | |||
=See aslo= | =See aslo= | ||
*[[Cas9]] | *[[Cas9]] | ||