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.