Protein Barcodes Enable High-Dimensional Single-Cell CRISPR Screens

Overview

Journal Cell

Publisher Cell Press

Specialty Cell Biology

Date 2018 Oct 23

PMID 30343902

Citations 72

Authors

Aleksandra Wroblewska

Maxime Dhainaut

Benjamin Ben-Zvi

Samuel A Rose

Eun Sook Park

El-Ad David Amir

Anela Bektesevic

Alessia Baccarini

Miriam Merad

Adeeb H Rahman

Brian D Brown

Affiliations

Soon will be listed here.

Abstract

CRISPR pools are being widely employed to identify gene functions. However, current technology, which utilizes DNA as barcodes, permits limited phenotyping and bulk-cell resolution. To enable novel screening capabilities, we developed a barcoding system operating at the protein level. We synthesized modules encoding triplet combinations of linear epitopes to generate >100 unique protein barcodes (Pro-Codes). Pro-Code-expressing vectors were introduced into cells and analyzed by CyTOF mass cytometry. Using just 14 antibodies, we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR, we simultaneously analyzed multiple phenotypic markers, including phospho-signaling, on dozens of knockouts. Pro-Code/CRISPR screens found two interferon-stimulated genes, the immunoproteasome component Psmb8 and a chaperone Rtp4, are important for antigen-dependent immune editing of cancer cells and identified Socs1 as a negative regulator of Pd-l1. The Pro-Code technology enables simultaneous high-dimensional protein-level phenotyping of 100s of genes with single-cell resolution.

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References

Pan D, Kobayashi A, Jiang P, Ferrari de Andrade L, Tay R, Luoma A . A major chromatin regulator determines resistance of tumor cells to T cell-mediated killing. Science. 2018; 359(6377):770-775. PMC: 5953516. DOI: 10.1126/science.aao1710. View

King S, Duggal N, Ndongmo C, Pacut C, Telesnitsky A . Pseudodiploid genome organization AIDS full-length human immunodeficiency virus type 1 DNA synthesis. J Virol. 2007; 82(5):2376-84. PMC: 2258930. DOI: 10.1128/JVI.02100-07. View

Amir E, Davis K, Tadmor M, Simonds E, Levine J, Bendall S . viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nat Biotechnol. 2013; 31(6):545-52. PMC: 4076922. DOI: 10.1038/nbt.2594. View

Jaitin D, Weiner A, Yofe I, Lara-Astiaso D, Keren-Shaul H, David E . Dissecting Immune Circuits by Linking CRISPR-Pooled Screens with Single-Cell RNA-Seq. Cell. 2016; 167(7):1883-1896.e15. DOI: 10.1016/j.cell.2016.11.039. View

Bassik M, Lebbink R, Churchman L, Ingolia N, Patena W, LeProust E . Rapid creation and quantitative monitoring of high coverage shRNA libraries. Nat Methods. 2009; 6(6):443-5. PMC: 2783737. DOI: 10.1038/nmeth.1330. View

Angelo M, Bendall S, Finck R, Hale M, Hitzman C, Borowsky A . Multiplexed ion beam imaging of human breast tumors. Nat Med. 2014; 20(4):436-42. PMC: 4110905. DOI: 10.1038/nm.3488. View

Bendall S, Simonds E, Qiu P, Amir E, Krutzik P, Finck R . Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science. 2011; 332(6030):687-96. PMC: 3273988. DOI: 10.1126/science.1198704. View

Adamson B, Norman T, Jost M, Cho M, Nunez J, Chen Y . A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response. Cell. 2016; 167(7):1867-1882.e21. PMC: 5315571. DOI: 10.1016/j.cell.2016.11.048. View

Minn A, Wherry E . Combination Cancer Therapies with Immune Checkpoint Blockade: Convergence on Interferon Signaling. Cell. 2016; 165(2):272-5. DOI: 10.1016/j.cell.2016.03.031. View

10.

Agudo J, Park E, Rose S, Alibo E, Sweeney R, Dhainaut M . Quiescent Tissue Stem Cells Evade Immune Surveillance. Immunity. 2018; 48(2):271-285.e5. PMC: 5824652. DOI: 10.1016/j.immuni.2018.02.001. View

11.

Cong L, Ran F, Cox D, Lin S, Barretto R, Habib N . Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339(6121):819-23. PMC: 3795411. DOI: 10.1126/science.1231143. View

12.

Schumacher T, Hacohen N . Neoantigens encoded in the cancer genome. Curr Opin Immunol. 2016; 41:98-103. DOI: 10.1016/j.coi.2016.07.005. View

13.

Peterson V, Zhang K, Kumar N, Wong J, Li L, Wilson D . Multiplexed quantification of proteins and transcripts in single cells. Nat Biotechnol. 2017; 35(10):936-939. DOI: 10.1038/nbt.3973. View

14.

Datlinger P, Rendeiro A, Schmidl C, Krausgruber T, Traxler P, Klughammer J . Pooled CRISPR screening with single-cell transcriptome readout. Nat Methods. 2017; 14(3):297-301. PMC: 5334791. DOI: 10.1038/nmeth.4177. View

15.

Sack L, Davoli T, Xu Q, Li M, Elledge S . Sources of Error in Mammalian Genetic Screens. G3 (Bethesda). 2016; 6(9):2781-90. PMC: 5015935. DOI: 10.1534/g3.116.030973. View

16.

Agudo J, Ruzo A, Park E, Sweeney R, Kana V, Wu M . GFP-specific CD8 T cells enable targeted cell depletion and visualization of T-cell interactions. Nat Biotechnol. 2015; 33(12):1287-1292. PMC: 4675673. DOI: 10.1038/nbt.3386. View

17.

Mullokandov G, Baccarini A, Ruzo A, Jayaprakash A, Tung N, Israelow B . High-throughput assessment of microRNA activity and function using microRNA sensor and decoy libraries. Nat Methods. 2012; 9(8):840-6. PMC: 3518396. DOI: 10.1038/nmeth.2078. View

18.

Shalem O, Sanjana N, Zhang F . High-throughput functional genomics using CRISPR-Cas9. Nat Rev Genet. 2015; 16(5):299-311. PMC: 4503232. DOI: 10.1038/nrg3899. View

19.

Doench J, Fusi N, Sullender M, Hegde M, Vaimberg E, Donovan K . Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016; 34(2):184-191. PMC: 4744125. DOI: 10.1038/nbt.3437. View

20.

Dixit A, Parnas O, Li B, Chen J, Fulco C, Jerby-Arnon L . Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens. Cell. 2016; 167(7):1853-1866.e17. PMC: 5181115. DOI: 10.1016/j.cell.2016.11.038. View