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bioRxiv · 10.1101/2025.04.28.650920

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

Kato, S.·Ikemoto, A.·Isayama, J.·Takimoto, T.·Saya, H.·Hamada, K.-i.
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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.

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Kato, S., Ikemoto, A., Isayama, J., Takimoto, T., Saya, H., Hamada, K.-i.. 2025-05-01. Proof-of-Concept of A DNA-Based Recording System for High-Throughput Functional Gene Screening. https://doi.org/10.1101/2025.04.28.650920

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