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

Abstract

Pooled genetic screening technologies offer high efficiency and enable systematic causal analyses. However, further reductions in cost and handling complexity are still desirable. Here we present PiER (Perturbation-induced Intracellular Events Recorder), a novel streamlined pooled genetic screening technology that couples gene perturbation with intracellular 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 site-specific recombinase when a chosen signaling pathway is activated; and a Memory domain whose sequence is permanently rewritten by the recombinase, storing perturbation-response histories in situ. In HEK293 cells, a WNT-responsive Response/Memory domain construct produced dose-dependent recombination signatures verified by a fluorescent reporter and quantitative PCR. Lentiviral delivery of a pooled shRNA PiER library subsequently identified WNT-related shRNA candidates. PiER thus provides a versatile, scalable tool for functional genomics and drug-target discovery.