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Expanding the range of Protospacer Adjacent Motifs (PAMs) recognized by CRISPR-Cas9 is essential for broadening genome-editing applications. Here, we combine molecular dynamics simulations with graph-theory and centrality analyses to dissect the principles of PAM recognition in three Cas9 variants - VQR, VRER, and EQR - that target non-canonical PAMs. We show that efficient recognition is not dictated solely by direct contacts between PAM-interacting residues and DNA, but also by a distal network that stabilizes the PAM-binding domain and preserves long-range communication with REC3, a hub that relays signals to the HNH nuclease. A key role emerges for the D1135V/E substitution, which enables stable DNA binding by K1107 and preserves key DNA phosphate locking interactions via S1109, securing stable PAM engagement. In contrast, variants carrying only R-to-Q substitutions at PAM-contacting residues, though predicted to enhance adenine recognition, destabilize the PAM-binding cleft, perturb REC3 dynamics, and disrupt allosteric coupling to HNH. Together, these findings establish that PAM recognition requires local stabilization, distal coupling, and entropic tuning, rather than a simple consequence of base-specific contacts. This framework provides guiding principles for engineering Cas9 variants with expanded PAM compatibility and improved editing efficiency. 

The revised, regularized Tao–Mo (rregTM) exchange-correlation density functional approximation (DFA) [A. Patra, S. Jana, and P. Samal, J. Chem. Phys. 153, 184112 (2020) and Jana et al., J. Chem. Phys. 155, 024103 (2021)] resolves the order-of-limits problem in the original TM formulation while preserving its valuable essential behaviors. Those include performance on standard thermochemistry and solid data sets that is competitive with that of the most widely explored meta-generalized-gradient-approximation DFAs (SCAN and r2SCAN) while also providing superior performance on elemental solid magnetization. Puzzlingly however, rregTM proved to be intractable for de-orbitalization via the approach of Mejía-Rodríguez and Trickey [Phys. Rev. A 96, 052512 (2017)]. We report investigation that leads to diagnosis of how the regularization in rregTM of the z indicator functions (z = the ratio of the von-Weizsäcker and Kohn–Sham kinetic energy densities) leads to non-physical behavior. We propose a simpler regularization that eliminates those oddities and that can be calibrated to reproduce the good error patterns of rregTM. We denote this version as simplified, regularized Tao–Mo, sregTM. We also show that it is unnecessary to use rregTM correlation with sregTM exchange: Perdew–Burke–Ernzerhof correlation is sufficient. The subsequent paper shows how sregTM enables some progress on de-orbitalization. 

In Paper I [H. Francisco, A. C. Cancio, and S. B. Trickey, J. Chem. Phys. 159, 214102 (2023)], we gave a regularization of the Tao–Mo exchange functional that removes the order-of-limits problem in the original Tao–Mo form and also eliminates the unphysical behavior introduced by an earlier regularization while essentially preserving compliance with the second-order gradient expansion. The resulting simplified, regularized (sregTM) functional delivers performance on standard molecular and solid state test sets equal to that of the earlier revised, regularized Tao–Mo functional. Here, we address de-orbitalization of that new sregTM into a pure density functional. We summarize the failures of the Mejía-Rodríguez and Trickey de-orbitalization strategy [Phys. Rev. A 96, 052512 (2017)] when used with both versions. We discuss how those failures apparently arise in the so-called z′ indicator function and in substitutes for the reduced density Laplacian in the parent functionals. Then, we show that the sregTM functional can be de-orbitalized somewhat well with a rather peculiarly parameterized version of the previously used deorbitalizer. We discuss, briefly, a de-orbitalization that works in the sense of reproducing error patterns but that apparently succeeds by cancelation of major qualitative errors associated with the de-orbitalized indicator functions α and z, hence, is not recommended. We suggest that the same issue underlies the earlier finding of comparatively mediocre performance of the de-orbitalized Tao–Perdew–Staroverov–Scuseri functional. Our work demonstrates that the intricacy of such two-indicator functionals magnifies the errors introduced by the Mejía-Rodríguez and Trickey de-orbitalization approach in ways that are extremely difficult to analyze and correct.