Fig. 6: Molecular modeling of the KG and VRKG variants’ altered PAM recognition.

a Schematic summary of the KG variant’s base-specific interactions with a NAG PAM. Major-groove interactions between R1335 and the non-target strand (non-TS) are maintained, as is the minor-groove interaction between K1107 and the target strand (TS). b Three-dimensional model of the KG variant’s PID interactions with a CAG PAM, based on a published crystal structure of PAM-bound Cas9⁵⁵ (PDB 5F9R). Relevant target strand nucleobases are labeled in parentheses. The D1332K, R1333G, (A1) → (G1), and (C2) → (T2) substitutions were introduced with PyMOL for visualization purposes. One of several K1332 rotamers in range for contacting the phosphate backbone is shown (dashed green line). c Schematics as in panel a, but focusing on K1107’s minor-groove interaction with the O2 acceptor of a TS pyrimidine (boxed inset, dashed gray lines). Thymine harbors more free electrons (curved dashed green line) at its O2 acceptor than cytosine when they are Watson–Crick paired in duplex DNA. d Schematics as in panel c, but summarizing a putative alternative interaction between K1107 and either (T1) or (C1) nucleobases at position 1 of the TS. e Three-dimensional model as in panel b, but for the VRKG variant. R1136 is in range for contacting G3’s acceptor group in the minor groove, without clashing with K1107. f Three-dimensional model as in panel e, but with an AAG PAM. When the alternative K1107 rotamer is adopted, R1136 may also adopt an alternative rotamer in range for minor-groove contacts with (T2) of the TS.