CRISPR activation and interference screens decode stimulation responses in primary human T cells

Ralf Schmidt; Zachary Steinhart; Madeline Layeghi; Jacob W Freimer; Raymund Bueno; Vinh Q Nguyen; Franziska Blaeschke; Chun Jimmie Ye; Alexander Marson

doi:10.1126/science.abj4008

CRISPR activation and interference screens decode stimulation responses in primary human T cells

Science. 2022 Feb 4;375(6580):eabj4008. doi: 10.1126/science.abj4008. Epub 2022 Feb 4.

Authors

Ralf Schmidt^#^{1

2}, Zachary Steinhart^#^{1

2}, Madeline Layeghi¹, Jacob W Freimer^{1

2

3}, Raymund Bueno², Vinh Q Nguyen⁴, Franziska Blaeschke^{1

2}, Chun Jimmie Ye^{1

2

5

6

7

8

9

10}, Alexander Marson^{1

2

5

11

12

13

14

6

7}

Affiliations

¹ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA.
² Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA.
³ Department of Genetics, Stanford University, Stanford, CA 94305, USA.
⁴ Department of Surgery, University of California San Francisco, San Francisco, CA 94143, USA.
⁵ Chan Zuckerberg Biohub, San Francisco, CA 94158, USA.
⁶ Parker Institute for Cancer Immunotherapy, University of California San Francisco, San Francisco, CA 94129, USA.
⁷ Institute for Human Genetics, University of California San Francisco, San Francisco, CA 94143, USA.
⁸ Department of Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, CA 94158, USA.
⁹ Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94158, USA.
¹⁰ Bakar Computational Health Sciences Institute, University of California, San Francisco, San Francisco, CA 94143, USA.
¹¹ Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA 94143, USA.
¹² Diabetes Center, University of California San Francisco, San Francisco, CA 94143, USA.
¹³ Innovative Genomics Institute, University of California Berkeley, Berkeley, CA 94720, USA.
¹⁴ UCSF Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94158, USA.

^# Contributed equally.

Abstract

Regulation of cytokine production in stimulated T cells can be disrupted in autoimmunity, immunodeficiencies, and cancer. Systematic discovery of stimulation-dependent cytokine regulators requires both loss-of-function and gain-of-function studies, which have been challenging in primary human cells. We now report genome-wide CRISPR activation (CRISPRa) and interference (CRISPRi) screens in primary human T cells to identify gene networks controlling interleukin-2 (IL-2) and interferon-γ (IFN-γ) production. Arrayed CRISPRa confirmed key hits and enabled multiplexed secretome characterization, revealing reshaped cytokine responses. Coupling CRISPRa screening with single-cell RNA sequencing enabled deep molecular characterization of screen hits, revealing how perturbations tuned T cell activation and promoted cell states characterized by distinct cytokine expression profiles. These screens reveal genes that reprogram critical immune cell functions, which could inform the design of immunotherapies.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

CRISPR-Associated Protein 9 / genetics
CRISPR-Associated Protein 9 / metabolism*
CRISPR-Cas Systems*
Cell Line
Cells, Cultured
Gene Expression Regulation
Gene Regulatory Networks*
Genome, Human
Humans
Interferon-gamma / biosynthesis*
Interferon-gamma / genetics
Interleukin-2 / biosynthesis*
Interleukin-2 / genetics
Lymphocyte Activation*
NF-kappa B / metabolism
RNA-Seq
Signal Transduction
Single-Cell Analysis
T-Lymphocytes / immunology*
T-Lymphocytes / metabolism

Substances

IL2 protein, human
Interleukin-2
NF-kappa B
Interferon-gamma
CRISPR-Associated Protein 9

Abstract

Publication types

MeSH terms

Substances

Grants and funding