Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9

Abstract

The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single turnover enzyme that displays a stable product state after double-stranded DNA cleavage. Here, we present cryo-EM structures of pre-catalytic, post-catalytic, and product states of the active Cas9-sgRNA-DNA complex in the presence of Mg2+. In the pre-catalytic state, Cas9 adopts the “checkpoint” conformation with the HNH nuclease domain positioned far away from the DNA. Transition to the post-catalytic state involves a dramatic ~34 Å swing of the HNH domain and disorder of the REC2 recognition domain. The post-catalytic state captures the cleaved substrate bound to the catalytically competent HNH active site. In the product state, the HNH domain is disordered, REC2 returns to the pre-catalytic conformation, and additional interactions of REC3 and RuvC with nucleic acids are formed. The coupled domain motions and interactions between the enzyme and nucleic acids provide new insights into the mechanism of genome editing by Cas9.

Quantification of Cas9 binding and cleavage across diverse guide sequences maps landscapes of target engagement

“…We speculate that P(stop) may be functionally equivalent to what we report as the probability of productive binding, and that nonproductive Cas9 RNP:target interactions are easily dismantled by either colliding with RNA polymerase or passing through nitrocelluose. Thus our work adds to the growing biochemical evidence of nonproductive bound states (12,13,33).…”

HK97 gp74 Possesses an α-Helical Insertion in the ββα Fold That Affects Its Metal Binding, cos Site Digestion, and In Vivo Activities

“…Note that dynamics have been demonstrated to control the activity of other HNH endonucleases, such as the Cas9 HNH domain (35,(54)(55)(56) and E9 colicin (57-59). Further, sequences outside the ␤␤␣ fold can impart specific functions to HNH proteins, such as binding to other proteins (25,44,60,61) or recognizing the nucleic acid substrate (22,25,34), and thereby provide specificity to HNH motif-containing proteins.…”

“…However, specific functions can be imparted to the HNH motif through additional structural elements. For example, the HNH motif is often part of larger multidomain proteins, such as the homing endonuclease I-HmuI (22), the restriction endonuclease EcoKMcrA (34), and CRISPR-Cas9 (31,(35)(36)(37). The additional domains perform other functions, such as DNA binding (e.g., I-HmuI and EcoKMcrA) or cutting one strand of the double-stranded DNA while the HNH domain cuts the other strand (e.g., the RuvC domain in Cas9).…”

Structure of a type IV CRISPR-Cas effector complex

“…In the multi-subunit class I systems, the CRISPR locus is transcribed and processed into small crRNA guides (CRISPR-derived RNA), around which several Cas proteins assemble to form large ribonucleoprotein complexes that facilitate RNA-guided surveillance and degradation of complementary targets 2 . While a myriad of structures have been determined for most types of CRISPR RNA-guided complexes (types I [3][4][5][6][7][8] , II [9][10][11] , III [12][13][14] , V [15][16][17] , VI [18][19][20], the RNA complexes of the highly diverse class 1 type IV CRISPR systems have largely remained structurally uncharacterised 1,21,22 . Type IV CRISPR systems occur within plasmid-like elements and lack genes encoding adaptation modules (Cas1, Cas2 and Cas4) 23,24 .…”