Decoupling deaminase recruitment reveals a cas association dependent origin of off-target base editing

Off-target editing is one of the most critical safety concerns in base editing for therapeutic applications. Understanding the biological origin of off-target activity is essential for designing safer base editors.

In this ASGCT 2026 poster, Revvity investigates the origins of off-target base editing using the Pin-point™ modular base editing platform. By independently controlling deaminase recruitment, the study demonstrates that both off-target editing and associated DNA damage are driven primarily by deaminase recruitment to Cas-bound DNA rather than free deaminase activity alone.

Two competing models of off-target editing were investigated:

• Cas-independent deaminase association: the deaminase acts on accessible single-stranded DNA (at replication forks or transcription bubbles) independently of the Cas component
• Cas-dependent deaminase association: transient interactions of the nCas component with PAM motifs during genome scanning drive deaminase recruitment and off-target editing
• Together, these data support a model in which deaminase recruitment kinetics are a key determinant of editing outcomes and suggest new approaches for improving the safety profile of therapeutic base editing programs.

Key findings:

• Off-target deamination depends on deaminase recruitment to Cas-bound DNA
• Whole-exome and genome sequencing analyses support a model in which transient Cas interactions during PAM scanning contribute to off-target editing
• DNA damage (γH2AX foci) requires aptamer-dependent deaminase recruitment; neither the deaminase nor Cas component alone produces the same effect
• Limiting deaminase recruitment reduces off-target editing and bystander editing while maintaining efficient on-target editing
• Modular base editors exhibit lower bystander editing than comparable fusion editors at equivalent on-target editing levels
• Controlling editor assembly, recruitment kinetics, and component stoichiometry provides a practical strategy to improve editing precision

The Pin-point™ base editing platform technology is available for clinical or diagnostic study and commercialization under a commercial license from Revvity.​