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

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.

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Figure 1: A high-throughput, image-based screening method using massively multiplexed fluorescence in situ hybridization.
Figure 2: Performance characterization of image-based screening.
Figure 3: Screening YFAST mutants for improved brightness and photostability.

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Acknowledgements

We thank H. Babcock for instrumentation advice and many readers for participating in the discussion of the preprint of this paper that we deposited on bioRxiv. This work was supported in part by the NIH. X.Z. is an HHMI investigator.

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Authors

Contributions

G.E., J.R.M., and X.Z. conceived the study and designed the experiments. G.E. performed experiments and analyzed data. G.E., J.R.M., and X.Z. interpreted the data and wrote the manuscript.

Corresponding authors

Correspondence to Jeffrey R Moffitt or Xiaowei Zhuang.

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Competing interests

G.E., J.R.M., and X.Z. are inventors on a patent applied for by Harvard University that covers the screening method described here.

Integrated supplementary information

Supplementary Figure 1 Distributions of the ratio of readout 0 intensity to the readout 1 intensity.

Histogram of the natural logarithm of the ratio of readout 0 intensity to readout 1 intensity for bit 1 for only those cells with assigned barcodes that match the valid barcodes that are determined to be in the library by sequencing. The histogram was fit to a sum of two skewed Gaussians and each fit Gaussian is depicted (green and magenta dashed lines). The overlap between the two Gaussian curves corresponds to 0.07% of the total number of cells presented here. The dotted cyan line depicts the bit-calling threshold. Cells above the bit-calling threshold were assigned a bit value of “0” for this bit while cells below the bit-calling threshold were assigned a bit value of “1”.

Supplementary Figure 2 Reversible and biphasic photobleaching kinetics of YFAST.

The normalized fluorescence intensity (green crosses) upon intermittent illumination with 488-nm light and a fit to a double exponential decay (purple line). E. coli expressing BFP-YFAST were adhered to a glass coverslip, immersed in 10 μM HMBR in PBS and imaged at 4-ms time resolution. The YFAST fluorescence intensity for each cell is normalized by the BFP fluorescence and averaged over multiple cells in the imaged area. The 488-nm illumination was switched on and off with a period of 2 seconds. Intensity values of zero represent the period of time when the illumination was off.

Supplementary Figure 3 Additional quantifications of the YFAST library screen and comparisons between library measurements and isolated mutant measurements.

(a) Scatter plot of the apparent fractional amplitudes of the fast bleaching component and the amplitudes of the slow bleaching component for different YFAST mutants in all of the libraries imaged. Each point depicts the median values of all cells associated with one mutant. The amplitudes of the slow bleaching component are normalized to that of the original YFAST. The apparent fractional amplitude of the fast bleaching component is defined as the amplitude of the fast bleaching component divided by the sum of the amplitudes of the fast and slow bleaching components. Here only the mutants that contain at least 10 imaged cells are depicted. The library measurements of the original YFAST and three selected mutants are indicated by the green star and colored circles, respectively. The color scheme is as in Fig. 3. (b) Scatter plot of the apparent fractional amplitudes of the fast bleaching component for each mutant measured in two replicate library measurements containing a subset of the 60,000 mutants. Each point represents the median of all cells corresponding to each mutant and is colored by the minimum number of cells measured in either replicate. Only the mutants with a slow component at least half as bright as the slow component for the original YFAST and that contain at least 10 imaged cells in each replicate are depicted. (c) The apparent fractional amplitude of the fast bleaching component for the original YFAST and the selected mutants determined from the isolated mutant measurements at the same time resolution as the library measurements shown together with the results obtained from the library measurements. Solid circles represent the median from the library measurements (N=420, 136, 32, 220 cells for the original YFAST, mutant 1, mutant 2, and mutant 3, respectively). Diamonds represent the mean from the isolated mutant measurements (N = 12, 5, 3, and 3 technical replicates for original YFAST, mutants 1, mutant 2, and mutant 3, respectively). Error bars represent SEM and individual measurements are depicted as gray crosses when N is less than 10. (d) The fluorescence intensity as a function of illumination time for the original YFAST and the three selected mutants (color-coded as in (a)) measured in isolation at the 4-ms time resolution. The fluorescence intensity is averaged over multiple cells. (e and f) The fractional amplitude (e) and photobleaching rate (f) of the fast bleaching component for the original YFAST and the selected mutants determined from the isolated mutant measurements shown in (d). Diamonds depict the mean and error bars represent SEM. Individual measurements are depicted as gray crosses (N = 3 replicates for the original YFAST and the three mutants). The amplitudes and rate constants of both fast and slow bleaching components are determined from these fast-time resolution measurements by a double exponential fit. The fractional amplitudes and the rate constants of the fast bleaching component are shown in the plot here, and the amplitudes and rate constants of the slow bleaching component are shown in Fig. 3f, g.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Tables 1–2 and Supplementary Note 1 (PDF 878 kb)

Life Sciences Reporting Summary (PDF 129 kb)

Supplementary Protocol

Library construction and imaging for high-throughput image-based screening using multiplexed FISH. (PDF 376 kb)

Supplementary Software

Software for analysis of image data and sequencing data. (ZIP 129 kb)

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Emanuel, G., Moffitt, J. & Zhuang, X. High-throughput, image-based screening of pooled genetic-variant libraries. Nat Methods 14, 1159–1162 (2017). https://doi.org/10.1038/nmeth.4495

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