High-throughput, image-based screening of pooled genetic-variant libraries

George Emanuel; Jeffrey R Moffitt; Xiaowei Zhuang

doi:10.1038/nmeth.4495

High-throughput, image-based screening of pooled genetic-variant libraries

Nat Methods. 2017 Dec;14(12):1159-1162. doi: 10.1038/nmeth.4495. Epub 2017 Oct 30.

Authors

George Emanuel^{1

2

3}, Jeffrey R Moffitt^{1

3}, Xiaowei Zhuang^{1

3}

Affiliations

¹ Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
² Graduate Program in Biophysics, Harvard University, Cambridge, Massachusetts, USA.
³ Department of Chemistry and Chemical Biology and Department of Physics, Harvard University, Cambridge, Massachusetts, USA.

Abstract

We report a high-throughput screening method that allows diverse genotypes and corresponding phenotypes to be imaged in individual cells. We achieve genotyping by introducing barcoded genetic variants into cells as pooled libraries and reading the barcodes out using massively multiplexed fluorescence in situ hybridization. To demonstrate the power of image-based pooled screening, we identified brighter and more photostable variants of the fluorescent protein YFAST among 60,000 variants.

MeSH terms

DNA Barcoding, Taxonomic / methods*
Escherichia coli / genetics*
Gene Library*
High-Throughput Nucleotide Sequencing / methods*
In Situ Hybridization, Fluorescence / methods*
Mutation
Reproducibility of Results

Grants and funding

R01 MH113094/MH/NIMH NIH HHS/United States