CRISPR-Cas9: Difference between revisions

SaCas9 recognizes the 5'-NNGRRN-3' PAM, with a preference for a thymine base at the 6th position, which is distinct from the 5'-NGG-3' PAM of SpCas9. In the present structures containing either the <scene name='74/742625/Cv8/3'>5'-TTGAAT-3' PAM</scene> or the <scene name='74/742625/Cv9/3'>5'-TTGGGT-3' PAM</scene>, the PAM duplex is sandwiched between the WED and PI domains, and the PAM in the non-target DNA strand is read from the major groove side by the PI domain. dT1* and dT2* do not directly contact the protein. Consistent with the observed requirement for the 3rd G in the 5'-NNGRRT-3' PAM, the O6 and N7 of dG3* form bidentate hydrogen bonds with the side chain of Arg1015, which is anchored via salt bridges with Glu993 in both complexes. In the 5'-TTGAAT-3' PAM complex, the <scene name='74/742625/Cv8/4'>N7 atoms of dA4* and dA5* form direct and water-mediated hydrogen bonds with Asn985 and Asn985/Asn986/Arg991</scene>, respectively. In addition, the N6 of dA5* forms a water-mediated hydrogen bond with Asn985. Similarly, in the 5'-TTGGGT-3' PAM complex, the <scene name='74/742625/Cv9/4'>N7 atoms of dG4* and dG5* form direct and water-mediated hydrogen bonds with Asn985 and Asn985/Asn986/Arg991</scene>, respectively. The O6 of dG5* forms a water-mediated hydrogen bond with Asn985. These structural features explain the ability of SaCas9 to recognize the purine nucleotides at positions 4 and 5 in the 5'-NNGRRT-3' PAM. The O4 of dT6* hydrogen bonds with Arg991, explaining the preference of SaCas9 for the 6th T in the 5'-NNGRRT-3' PAM. Single alanine mutations of these PAM-interacting residues reduced the cleavage activity in vivo, and double mutations abolished the activity, confirming the importance of Asn985, Asn986, Arg991, Glu993, and Arg1015 for PAM recognition. In addition, <scene name='74/742625/Cv8/6'>the phosphate backbone of the PAM duplex is recognized from the minor groove side by the WED domain (Tyr789, Tyr882, Lys886, Ans888, Ala889, and Leu909)</scene>, in a distinct manner from that in SpCas9. Together, these structural and functional data have revealed the mechanism underlying the relaxed recognition of the 5'-NNGRRT-3' PAM by SaCas9.