FIGURE 6.

Conformational basis of off-target effects. (a) Crystal structure of CRISPR-Cas9 in a “conformational checkpoint” state (PDB code: 4UN3).¹⁰ The arrow indicates the conformational change required by the inactive HNH to cleave the DNA target strand (TS). (b) Extended opening of the RNA:DNA hybrid and newly formed interactions with the L2 loop (magenta), observed during MD simulations of CRISPR-Cas9 in the presence of four base pair mismatches at PAM distal sites. The 692–700 α-helix of the Rec3 region (black) is shown to insert within the within the RNA:DNA, promoting its extended opening. (c) RNA:DNA minor groove width computed along MD simulations of CRISPR-Cas9 bound to an on-target DNA (black) and in the presence of one to four PAM distal mismatches (left panel). A vertical bar indicates the experimental minor groove width (i.e., 11 Å from x-ray crystallography). The minor groove width has been measured at the level of base pair 17 (right panel).¹⁹ Reprinted with permission from Reference 19 Copyright 2019 American Chemical Society. https://pubs.acs.org/doi/full/10.1021/acscentsci.9b00020. Further permissions related to the material excerpted should be directed to the American Chemical Society