The journal of physical chemistry. BNov 11, 2025

How to Broaden DNA Targeting Options in CRISPR-Cas9 Using VQR, VRER, and EQR Variants

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jan 3, 2026

Abstract

Efficient recognition of noncanonical PAMs by Cas9 variants is influenced by a distal network that stabilizes the PAM-binding domain.

PAM recognition involves both direct interactions with DNA and a distal network that maintains long-range communication with the HNH nuclease.
The D1135 V/E substitution is crucial for stable DNA binding and enhances PAM engagement through interactions with K1107 and S1109.
Variants with only R-to-Q substitutions at PAM-contacting residues may destabilize the PAM-binding cleft and disrupt communication with REC3.
Recognition of PAMs is associated with local stabilization and entropic tuning, rather than being solely based on base-specific contacts.
These findings offer insights for engineering Cas9 variants that could broaden PAM compatibility and improve genome-editing efficiency.

Simplified

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 noncanonical 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 D1135 V/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.

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How to Broaden DNA Targeting Options in CRISPR-Cas9 Using VQR, VRER, and EQR Variants

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