Extended Data Fig. 1. Characterization of the Cas9:DNA cross-link.

a, Crystal structure of Cas9:sgRNA:DNA with 20-bp RNA:DNA hybrid formed (PDB 4UN3). In the inset, Arg1333 and Arg1335 recognize the two guanines of the PAM. Green, NUC lobe; blue, REC lobe; orange, guide RNA; black, DNA; yellow, PAM. b, Non-reducing SDS-PAGE (Stain-Free) analysis of cross-linking reactions and controls. Complexes were prepared identically to structural constructs but in smaller volumes and without size exclusion purification. Competitor duplex, where indicated, was added before the cross-linkable duplex at an equivalent concentration. The depicted experiment was performed once, and a prior optimization experiment yielded similar results. c, Top left, non-reducing SDS-PAGE autoradiograph to determine the fraction of DNA cross-linked to Cas9 at t=0. The target strand is radiolabeled. Bottom, reducing urea-PAGE autoradiograph revealing target-strand cleavage kinetics; quantification depicted in top right. The depicted model is $y=C\left(1-e^{-k_{1}t}\right)+(B_{max}-C)(1-e^{-k_{2}t})$. The depicted experiments were performed once, and a prior optimization experiment yielded similar results.