Proof-of-Concept of a DNA-Based Recording System for High-Throughput Functional Gene Screening

Pooled genetic screening enables systematic causal analyses, but a lower cost and simpler handling remain desirable. Here we present PiER (Perturbation-induced Intracellular Events Recorder), a pooled screening technology that couples gene perturbation with signal recording and does not require single-cell isolation, cell sorting, or survival selection. PiER consists of three DNA domains: a Perturbation domain that introduces gene-specific perturbations; a Response domain that expresses a recombinase upon pathway activation; and a Recorder domain whose sequence is permanently rewritten by the recombinase, storing perturbation-response histories in situ. This architecture enables the next-generation sequencing readout of perturbations and response histories from bulk DNA. In HEK293 cells, a WNT-responsive Response/Recorder construct produced dose-dependent recombination detected by a fluorescent reporter and qPCR, and a second cAMP/CREB-responsive pilot vector showed stimulus-dependent recombination. Using the lentiviral delivery of a pooled shRNA PiER library, we identified WNT-related candidates. PiER provides a versatile, scalable tool for functional genomics and drug-target discovery.