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A CRISPR screen identifies a pathway required for paraquat-induced cell death

Abstract

Paraquat, a herbicide linked to Parkinson's disease, generates reactive oxygen species (ROS), which causes cell death. Because the source of paraquat-induced ROS production remains unknown, we conducted a CRISPR-based positive-selection screen to identify metabolic genes essential for paraquat-induced cell death. Our screen uncovered three genes, POR (cytochrome P450 oxidoreductase), ATP7A (copper transporter), and SLC45A4 (sucrose transporter), required for paraquat–induced cell death. Furthermore, our results revealed POR as the source of paraquat-induced ROS production. Thus, our study highlights the use of functional genomic screens for uncovering redox biology.

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Figure 1: POR, ATP7A, and SLC45A4 are essential for paraquat-induced cell death.
Figure 2: POR is necessary for paraquat-induced cytosolic ROS generation.
Figure 3: Mitochondrial complex I is not necessary for paraquat-induced cell death.
Figure 4: Paraquat negative-selection CRISPR-based screen.

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Acknowledgements

We thank the Genome Technology Core of the Whitehead Institute for deep sequencing the genomic DNA samples. We thank the Flow Cytometry Core of Northwestern University for providing the BD LSRFortessa cell analyzer to assess cell viability. We are grateful to M.P. Murphy (University of Cambridge) for providing us MitoParaquat. This work was supported by a National Institute of Aging grant (5P01AG049665) to N.S.C. C.R.R. was supported by a National Institutes of Health postdoctoral training grant (T32 HL076139-11). H.K. was supported by a National Institutes of Health pre-doctoral training grant (T32 CA9560-30). K.B. was supported by grants from the National Institutes of Health (K22 CA1936600), a Searle Scholar Award, an Irma T. Hirschl/Monique Weill–Caulier Trust Award, and the Sidney Kimmel Foundation. D.M.S. is an investigator of the Howard Hughes Medical Institute.

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Contributions

C.R.R., K.B., D.M.S., and N.S.C. initiated the project and designed the research plan. C.R.R. and K.B. conducted the PQ positive- and negative-selection CRISPR-based screens. T.W. performed the bioinformatics analysis of the deep sequencing data. C.R.R. and H.K. generated clonal knockout and cDNA overexpression Jurkat and A549 cell lines. C.R.R. and H.K. prepared genomic DNA for sequencing the knockout clones. H.K. performed all western blot analyses and the A549 viability assays. C.R.R. assessed Jurkat cell viability. I.M.-R. performed the ROS measurements using CM-H2DCFDA and Amplex red and assessed SOD1 activity. P.G. performed the mass spectrometry experiments to assess PQ uptake. C.R.R. and N.S.C. wrote the manuscript, with input from all authors.

Corresponding author

Correspondence to Navdeep S Chandel.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Tables 1–2, Supplementary Figures 1–10 (PDF 22728 kb)

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Reczek, C., Birsoy, K., Kong, H. et al. A CRISPR screen identifies a pathway required for paraquat-induced cell death. Nat Chem Biol 13, 1274–1279 (2017). https://doi.org/10.1038/nchembio.2499

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