A multiplexed epitope barcoding strategy that enables dynamic cellular phenotypic screens

Takamasa Kudo, Keara Lane, Markus W. Covert*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

Pooled genetic libraries have improved screening throughput for mapping genotypes to phenotypes. However, selectable phenotypes are limited, restricting screening to outcomes with a low spatiotemporal resolution. Here, we integrated live-cell imaging with pooled library-based screening. To enable intracellular multiplexing, we developed a method called EPICode that uses a combination of short epitopes, which can also appear in various subcellular locations. EPICode thus enables the use of live-cell microscopy to characterize a phenotype of interest over time, including after sequential stimulatory/inhibitory manipulations, and directly connects behavior to the cellular genotype. To test EPICode's capacity against an important milestone—engineering and optimizing dynamic, live-cell reporters—we developed a live-cell PKA kinase translocation reporter with improved sensitivity and specificity. The use of epitopes as fluorescent barcodes introduces a scalable strategy for high-throughput screening broadly applicable to protein engineering and drug discovery settings where image-based phenotyping is desired.

Original languageEnglish (US)
Pages (from-to)376-387.e8
JournalCell Systems
Volume13
Issue number5
DOIs
StatePublished - May 18 2022

Funding

We thank members of the Covert lab for discussions and for providing feedback on the manuscript. We gratefully acknowledge funding from The Paul G. Allen Frontiers Group Allen Discovery Center grant to M.W.C. and the Nakajima Foundation Scholarship to T.K.

Keywords

  • epitope barcodes
  • fluorescent reporters
  • in situ genotyping
  • pooled screen
  • spatial multiplexing

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology

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