A CRISPR-Cas9 delivery system for in vivo screening of genes in the immune system

Martin W LaFleur; Thao H Nguyen; Matthew A Coxe; Kathleen B Yates; Justin D Trombley; Sarah A Weiss; Flavian D Brown; Jacob E Gillis; Daniel J Coxe; John G Doench; W Nicholas Haining; Arlene H Sharpe

doi:10.1038/s41467-019-09656-2

A CRISPR-Cas9 delivery system for in vivo screening of genes in the immune system

Nat Commun. 2019 Apr 10;10(1):1668. doi: 10.1038/s41467-019-09656-2.

Authors

Martin W LaFleur^{1

2

3}, Thao H Nguyen^{1

3}, Matthew A Coxe^{1

3}, Kathleen B Yates^{2

4}, Justin D Trombley^{1

3}, Sarah A Weiss², Flavian D Brown^{1

2

3}, Jacob E Gillis^{1

3}, Daniel J Coxe⁵, John G Doench⁴, W Nicholas Haining^{6

7}, Arlene H Sharpe^{8

9

10}

Affiliations

¹ Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, 02115, USA.
² Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, 02115, USA.
³ Evergrande Center for Immunological Diseases, Harvard Medical School and Brigham and Women's Hospital, Boston, MA, 02115, USA.
⁴ Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, 02142, USA.
⁵ School of Energy, Matter, and Transport Engineering at Arizona State University, Tempe, AZ, 85287, USA.
⁶ Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, 02115, USA. Nicholas_Haining@dfci.harvard.edu.
⁷ Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, 02142, USA. Nicholas_Haining@dfci.harvard.edu.
⁸ Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, 02115, USA. Arlene_Sharpe@hms.harvard.edu.
⁹ Evergrande Center for Immunological Diseases, Harvard Medical School and Brigham and Women's Hospital, Boston, MA, 02115, USA. Arlene_Sharpe@hms.harvard.edu.
¹⁰ Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, 02142, USA. Arlene_Sharpe@hms.harvard.edu.

Abstract

Therapies that target the function of immune cells have significant clinical efficacy in diseases such as cancer and autoimmunity. Although functional genomics has accelerated therapeutic target discovery in cancer, its use in primary immune cells is limited because vector delivery is inefficient and can perturb cell states. Here we describe CHIME: CHimeric IMmune Editing, a CRISPR-Cas9 bone marrow delivery system to rapidly evaluate gene function in innate and adaptive immune cells in vivo without ex vivo manipulation of these mature lineages. This approach enables efficient deletion of genes of interest in major immune lineages without altering their development or function. We use this approach to perform an in vivo pooled genetic screen and identify Ptpn2 as a negative regulator of CD8⁺ T cell-mediated responses to LCMV Clone 13 viral infection. These findings indicate that this genetic platform can enable rapid target discovery through pooled screening in immune cells in vivo.

Publication types

Research Support, N.I.H., Intramural
Research Support, Non-U.S. Gov't

MeSH terms

Adaptive Immunity / genetics*
Animals
Bone Marrow Transplantation
CD8-Positive T-Lymphocytes / immunology
CRISPR-Cas Systems / genetics*
Cell Line, Tumor
Cell Lineage / genetics
Cell Lineage / immunology
Chlorocebus aethiops
Disease Models, Animal
Feasibility Studies
Female
Gene Transfer Techniques*
Genetic Testing / methods*
Genetic Vectors / genetics
Genomics / methods
HEK293 Cells
Humans
Immunity, Innate / genetics*
Lymphocytic Choriomeningitis / genetics
Lymphocytic Choriomeningitis / immunology
Lymphocytic Choriomeningitis / virology
Lymphocytic choriomeningitis virus / immunology
Male
Mice
Mice, Inbred C57BL
Mice, Transgenic
Protein Tyrosine Phosphatase, Non-Receptor Type 2 / genetics
Protein Tyrosine Phosphatase, Non-Receptor Type 2 / immunology
RNA, Guide, CRISPR-Cas Systems / genetics
Transplantation Chimera
Vero Cells

Substances

RNA, Guide, CRISPR-Cas Systems
PTPN2 protein, human
Protein Tyrosine Phosphatase, Non-Receptor Type 2

Abstract

Publication types

MeSH terms

Substances

Grants and funding