Sentinel cells with permanent memory via synthetic receptor-DNA typewriter integration

Current synthetic receptor systems generate transient outputs that vanish within hours or days, leaving no record of what cells sensed or when. This fundamentally limits their use in applications requiring long-term monitoring and recording temporal dynamics. This is unfortunate, since engineered sentinel cells otherwise represent a promising platform for biosensing, diagnostics, and cellular therapy applications. Their autonomous, programmable, and intrinsically biocompatible nature makes them ideal candidates for monitoring complex biological environments.

Here, we present a synNotch DNA typewriter that combines highly programmable synthetic Notch receptors with a CRISPR-Cas9 prime-editing-based technology called DNA typewriter. This combined system enables engineered cells to sense arbitrary input signals and permanently store this information in synthetic DNA tape, allowing post-hoc recovery of event history through sequencing.

Our design integrates three components: synNotch receptors, the DNA typewriter, and signal-controlled pegRNA expression via ENGRAM. SynNotch receptors are modular derivatives of native Notch, with swappable extracellular recognition and intracellular effector domains enabling programmable input-output coupling.

The DNA typewriter consists of tandem repeats of 5'-truncated CRISPR-Cas9 guide sequences, where only the first repeat is complete and targetable by a prime editor. Upon insertion of a designed barcode followed by a key sequence, the initial guide is disrupted while simultaneously completing the next guide. This creates sequential editing from first to last position, with event history recoverable from the order of barcode insertions.

To couple synNotch activation to prime editing, we placed pegRNAs under synNotch-controlled promoters using the ENGRAM system. In ENGRAM, the pegRNA is flanked by Csy4 hairpins within a transcript encoding the Csy4 endoribonuclease. Upon translation, Csy4 localizes to the nucleus and cleaves its cognate hairpins, releasing mature pegRNA. This architecture enables multiplex recording by matching orthogonal synNotch transcription factors to distinct ENGRAM-pegRNA cassettes.

We validated this system in engineered HEK293T cells with stable integration of TAPE, an anti-GFP synNotch receptor, and the corresponding ENGRAM cassette. The prime editor was delivered by transient transfection. RT-qPCR confirmed signal-dependent pegRNA expression, and Illumina sequencing demonstrated permanent recording of receptor activation in the TAPE locus, which matched well with the activation of a classic fluorescence-based synNotch reporter.

This work establishes a foundation for sentinel cells with permanent molecular memory. Potential applications include tracking immune cell dynamics during immunotherapy, recording developmental signalling, and multiplexed biosensing through orthogonal receptor-pegRNA pairs. The integration of synthetic receptors with DNA recording technology opens new possibilities for understanding and engineering cellular behaviour in complex biological systems.