CRISPR-Cas9: Difference between revisions

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Alexander Berchansky, Michal Harel

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 '''Structure of the sgRNA–Target DNA Complex'''
-The SaCas9 sgRNA consists of the guide region (G1–C20),repeat region (G21–G34), tetraloop (G35–A38), anti-repeat region (C39–C54), stem loop 1 (A56–G68), and single-stranded linker (U69–U73), with A55 connecting the anti-repeat region and stem loop 1. U73 at the 3' end is disordered in the present structure. The guide region (G1–C20) and the target DNA strand (dG1–dC20) form the <scene name='74/742625/Cv6/11'>guide:target heteroduplex</scene>, whereas the target DNA strand (dC(8)–dA(1)) and the non-target DNA strand (dT1*–dG8*) form a <scene name='74/742625/Cv6/12'>PAM-containing duplex</scene> (referred to as the PAM duplex). The repeat (G21–G34) and anti-repeat (C39–C54) regions form a distorted duplex (referred to as the <scene name='74/742625/Cv6/13'>repeat:anti-repeat duplex</scene>) via 13 Watson-Crick base pairs.
+The SaCas9 sgRNA consists of the guide region (G1–C20),repeat region (G21–G34), tetraloop (G35–A38), anti-repeat region (C39–C54), stem loop 1 (A56–G68), and single-stranded linker (U69–U73), with A55 connecting the anti-repeat region and stem loop 1. U73 at the 3' end is disordered in the present structure. The guide region (G1–C20) and the target DNA strand (dG1–dC20) form the <scene name='74/742625/Cv6/11'>guide:target heteroduplex</scene>, whereas the target DNA strand (dC(8)–dA(1)) and the non-target DNA strand (dT1*–dG8*) form a <scene name='74/742625/Cv6/12'>PAM-containing duplex</scene> (referred to as the PAM duplex). The repeat (G21–G34) and anti-repeat (C39–C54) regions form a distorted duplex (referred to as the <scene name='74/742625/Cv6/13'>repeat:anti-repeat duplex</scene>) via 13 Watson-Crick base pairs. <scene name='74/742625/Cv6/16'>Stem loop 1</scene> is formed via three Watson-Crick base pairs (G57:C67–C59:G65) and two non-canonical base pairs (A56:G68 and A60:A63). U64 does not base pair with A60 and is flipped out of the stem loop. The N1 and N6 of A63 hydrogen bond with the 2'-OH and N3 of A60, respectively. G68 stacks with G57:C67, with the G68 N2 interacting with the backbone phosphate group between A55 and A56. A55 adopts the ''syn'' conformation, and its adenine base stacks with U69. In addition, the N1 of A55 hydrogen bonds with the 2'-OH of G68, thus stabilizing the basal region of stem loop 1. An adenosine residue immediately after the repeat:anti-repeat duplex is highly conserved among CRISPR-Cas9 systems, and the equivalent adenosine in the SpCas9 sgRNA, A51, also adopts the ''syn'' conformation, suggesting that these adenosine residues play conserved key roles in connecting the repeat:anti-repeat duplex and stem loop 1.
 =See aslo=
 *[[Cas9]]