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Functional genomics

Shining a light on genetic screen strategies

Nature Reviews Genetics volume 19, pages 6–7 (2018)Cite this article

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Genetic screens in cultured cells are powerful methods for testing libraries of constructs (such as variants of one or more genes, or reagents to perturb the expression of many genes genome wide) for those that affect a phenotype of interest. These screens have typically been in one of two formats, each with distinct benefits and limitations. In arrayed, well-by-well screens, a single known variant or perturbation is applied to the multiple cells in each well. This allows the monitoring of sophisticated cellular phenotypes through high-content microscopy, but the cost and space of arrayed-format reagents can limit the size of the screened library. In pooled screens, a library is applied en masse to a large population of cells. Following the bulk selection of cells of interest (such as through drug selection or sorting based on a fluorescent reporter), the relevant constructs are identified by sequencing. Despite the higher throughput of pooled screens, they are limited by the relatively simplistic phenotypes that are probed via the selection step.

Emanuel et al. sought an approach whereby complex phenotypes could be probed by microscopy in pooled screens. The main technical hurdle is devising a strategy to deduce the construct received by each cell in situ in the absence of the well identity of arrayed screens. For their experimental system they used Escherichia coli cells and a library of ~60,000 variants of the fluorescent protein YFAST. To allow variant identification, each variant construct also expressed a barcode that serves as a hybridization target for the multiplexed error-robust fluorescence in situ hybridization (MERFISH) technique, which was previously established in the authors' laboratory. In the current study, MERFISH was adapted to identify the single type of expressed barcode per cell rather than to identify multiple endogenous transcripts per cell as for previous uses.

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References

  1. Emanuel, G., Moffitt, J. R. & Zhuang, X. High-throughput, image-based screening of pooled genetic-variant libraries. Nat. Methods http://dx.doi.org/10.1038/nmeth.4495 (2017)

  2. Lawson, M. J. et al. In situ genotyping of a pooled strain library after characterizing complex phenotypes. Mol. Syst. Biol. 13, 947 (2017)10.15252/msb.20177951

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  1. Darren J. Burgess
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Burgess, D. Shining a light on genetic screen strategies. Nat Rev Genet 19, 6–7 (2018). https://doi.org/10.1038/nrg.2017.99

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