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Updated: Jan 26, 2026
Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines
Published on: November 13, 2021
A CRISPR-Cas9 delivery system for in vivo screening of genes in the immune system
Martin W LaFleur1,2,3, Thao H Nguyen1,3, Matthew A Coxe1,3
1Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, 02115, USA.
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.
Insights
A new CRISPR-Cas9 system, CHimeric IMmune Editing (CHIME), enables rapid gene function evaluation in immune cells in vivo. This method identified Ptpn2 as a key regulator of CD8+ T cell responses.
Area of Science:
- Immunology
- Genomics
- Gene Editing
Background:
- Targeting immune cell function is crucial for treating cancer and autoimmune diseases.
- Functional genomics in primary immune cells is challenging due to inefficient vector delivery and cell state perturbation.
- Existing methods limit the study of gene function in mature immune cell lineages in vivo.
Purpose of the Study:
- To develop a novel CRISPR-Cas9 delivery system for in vivo functional genomics in immune cells.
- To enable rapid evaluation of gene function in innate and adaptive immune cells without ex vivo manipulation.
- To identify novel therapeutic targets for immune-mediated diseases.
Main Methods:
- Developed CHimeric IMmune Editing (CHIME), a CRISPR-Cas9 bone marrow delivery system.
- Utilized CHIME for efficient in vivo gene deletion in major immune lineages without affecting cell development or function.
- Performed an in vivo pooled genetic screen to identify genes regulating immune cell responses.
Main Results:
- CHIME allows for efficient and precise gene editing in vivo in major immune cell types.
- The study successfully identified Ptpn2 as a negative regulator of CD8+ T cell responses.
- Demonstrated the utility of CHIME in a pooled genetic screen for immune cell function.
Conclusions:
- CHIME is a powerful genetic platform for rapid target discovery through in vivo pooled screening in immune cells.
- This technology overcomes limitations of previous functional genomics approaches in primary immune cells.
- The findings highlight Ptpn2 as a potential therapeutic target for enhancing CD8+ T cell-mediated immunity.
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