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CRISPR/Cas9-Based Genetic Screening to Study T-Cell Function

  • Wanjing Shang
  • Fei Wang
  • Qi Zhu
  • Liangyu Wang
  • Haopeng WangEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2111)

Abstract

T-cell-based cancer immunotherapies have emerged as a promising approach for cancer treatment, highlighting the importance of understanding the regulation of T-cell function. However, the molecular mechanisms underlying T-cell activation are not fully understood. The CRISPR/Cas9 system can serve as a robust method to systematically study signaling pathways. In this chapter, we describe details of using the CRISPR screen to identify regulators in TCR signaling, from the sgRNA library construction to genomic DNA sequencing. We also add some notes to further help readers performing the CRISPR screen. This approach can be readily adapted to study the activation of other immune cells, including B cells and dendritic cells.

Key words

gRNA library T-cell activation Lentivirus production and titer Lentiviral transduction Cell sorting Genomic DNA extraction 

Notes

Acknowledgments

The authors thank F. Wang, Z. Lin (ShanghaiTech University), and J. M. Shen (The Semiconductor Manufacturing International Corporation Private School) for their critical reading of the manuscript. H.W. is funded by National Natural Science Foundation of China Grant 31670919 as well as the 1,000-Youth Elite Program of China.

References

  1. 1.
    Courtney AH, Lo WL, Weiss A (2018) TCR signaling: mechanisms of initiation and propagation. Trends Biochem Sci 43(2):108–123CrossRefGoogle Scholar
  2. 2.
    Wang H et al (2010) ZAP-70: an essential kinase in T-cell signaling. Cold Spring Harb Perspect Biol 2(5):a002279CrossRefGoogle Scholar
  3. 3.
    Zhou P et al (2014) In vivo discovery of immunotherapy targets in the tumour microenvironment. Nature 506(7486):52–57CrossRefGoogle Scholar
  4. 4.
    Boettcher M, McManus MT (2015) Choosing the right tool for the job: RNAi, TALEN, or CRISPR. Mol Cell 58(4):575–585CrossRefGoogle Scholar
  5. 5.
    Shang W et al (2018) Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation. Proc Natl Acad Sci U S A 115(17):E4051–E4060CrossRefGoogle Scholar
  6. 6.
    Shifrut E et al (2018) Genome-wide CRISPR screens in primary human T cells reveal key regulators of immune function. Cell 175(7):1958–1971.e1915CrossRefGoogle Scholar
  7. 7.
    Chi S, Weiss A, Wang H (2016) A CRISPR-based toolbox for studying T cell signal transduction. Biomed Res Int 2016:5052369PubMedPubMedCentralGoogle Scholar
  8. 8.
    Shang W, Wang F, Fan G, Wang H (2017) Key elements for designing and performing a CRISPR/Cas9-based genetic screen. J Genet Genomics 44(9):439–449CrossRefGoogle Scholar
  9. 9.
    Boettcher M et al (2019) Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding. BMC Genomics 20(1):107CrossRefGoogle Scholar
  10. 10.
    Zhu S et al (2019) Guide RNAs with embedded barcodes boost CRISPR-pooled screens. Genome Biol 20(1):20CrossRefGoogle Scholar
  11. 11.
    Tian R et al (2015) Combinatorial proteomic analysis of intercellular signaling applied to the CD28 T-cell costimulatory receptor. Proc Natl Acad Sci U S A 112(13):E1594–E1603CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Wanjing Shang
    • 1
    • 2
    • 3
  • Fei Wang
    • 1
  • Qi Zhu
    • 1
  • Liangyu Wang
    • 1
  • Haopeng Wang
    • 1
    Email author
  1. 1.School of Life Science and TechnologyShanghaiTech UniversityShanghaiChina
  2. 2.Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological SciencesChinese Academy of SciencesShanghaiChina
  3. 3.University of Chinese Academy of SciencesBeijingChina

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