HDR on Demand: Cell-Cycle–Synchronized, Pulse-Delivered Cas9 RNPs with Adaptive Donor Release

Develop a closed-loop editing workflow that senses cell-cycle state (e.g., FUCCI or CDK reporters) and delivers brief pulses of Cas9 RNPs plus donor templates during S/G2, optionally combined with transient HDR-biasing small molecules. Implement via optimized nucleofection for primary cells or via nanoparticles with S/G2-responsive release chemistry. This approach integrates precise RNP delivery with HDR steering and therapeutic precision goals, adapting non-viral vectors for state-triggered release. Timing payload delivery to cell-cycle state should increase knock-in rates, reduce indel burden, and limit cytotoxicity, providing a generalizable protocol for high-fidelity knock-ins across challenging cell types.

References:

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