bioRxiv : the preprint server for biologySep 15, 2025

Design rules for increasing DNA target range in CRISPR-Cas9 using VQR, VRER, and EQR variants

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jan 3, 2026

Abstract

Efficient recognition of non-canonical PAMs by Cas9 variants is influenced by a complex network rather than just direct DNA contacts.

Three Cas9 variants (VQR, VRER, and EQR) were analyzed for their PAM recognition capabilities.
A distal network stabilizes the PAM-binding domain and maintains long-range communication with REC3, which is crucial for signal relay to the HNH nuclease.
The D1135V/E substitution plays a significant role in solidifying DNA binding and maintaining essential interactions for stable PAM engagement.
Variants with only R-to-Q substitutions at PAM-contacting sites may enhance adenine recognition but disrupt PAM-binding stability and REC3 dynamics.
PAM recognition involves local stabilization and entropic tuning, highlighting a more intricate mechanism than previously understood.

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 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.

Funding

Competing interests

The authors declare no competing interests.

PubMed

Design rules for increasing DNA target range in CRISPR-Cas9 using VQR, VRER, and EQR variants

Abstract

Funding

Competing interests

You found one interesting study. We’ll send the next 7.

What Lands in Your Inbox Each Week:

Recent issues from the crispr gene editing brief

Abstract

Funding

Competing interests

Related papers

You found one interesting study. We’ll send the next 7.

What Lands in Your Inbox Each Week:

Recent issues from the crispr gene editing brief