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Updated: Nov 19, 2025
In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity
Published on: November 2, 2020
In vivo screens using a selective CRISPR antigen removal lentiviral vector system reveal immune dependencies in renal
Juan Dubrot1, Sarah Kate Lane-Reticker1, Emily A Kessler1
1Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, USA.
Abstract:
CRISPR-Cas9 genome engineering has increased the pace of discovery for immunology and cancer biology, revealing potential therapeutic targets and providing insight into mechanisms underlying resistance to immunotherapy. However, endogenous immune recognition of Cas9 has limited the applicability of CRISPR technologies in vivo. Here, we characterized immune responses against Cas9 and other expressed CRISPR vector components that cause antigen-specific tumor rejection in several mouse cancer models. To avoid unwanted immune recognition, we designed a lentiviral vector system that allowed selective CRISPR antigen removal (SCAR) from tumor cells. The SCAR system reversed immune-mediated rejection of CRISPR-modified tumor cells in vivo and enabled high-throughput genetic screens in previously intractable models. A pooled in vivo screen using SCAR in a CRISPR-antigen-sensitive renal cell carcinoma revealed resistance pathways associated with autophagy and major histocompatibility complex class I (MHC class I) expression. Thus, SCAR presents a resource that enables CRISPR-based studies of tumor-immune interactions and prevents unwanted immune recognition of genetically engineered cells, with implications for clinical applications.
Insights
CRISPR-Cas9 immune responses limit in vivo applications. A new Selective CRISPR Antigen Removal (SCAR) system overcomes this, enabling tumor immune interaction studies and therapeutic development.
Area of Science:
- Immunology
- Cancer Biology
- Genome Engineering
Background:
- CRISPR-Cas9 advances immunology and cancer research but faces limitations due to immune recognition of Cas9 in vivo.
- Endogenous immune responses against CRISPR components can cause antigen-specific tumor rejection, hindering therapeutic applications.
Purpose of the Study:
- To characterize immune responses against CRISPR-Cas9 components.
- To develop a system to overcome immune rejection of CRISPR-modified cells.
- To enable high-throughput genetic screens in immune-sensitive cancer models.
Main Methods:
- Characterization of immune responses against Cas9 and CRISPR vector components in mouse cancer models.
- Design and implementation of a lentiviral vector system for Selective CRISPR Antigen Removal (SCAR) from tumor cells.
- In vivo pooled genetic screens using the SCAR system in a CRISPR-antigen-sensitive renal cell carcinoma model.
Main Results:
- Immune recognition of Cas9 leads to antigen-specific tumor rejection in vivo.
- The SCAR system effectively reversed immune-mediated rejection of CRISPR-modified tumor cells.
- SCAR enabled high-throughput genetic screens in previously intractable models, revealing resistance pathways involving autophagy and MHC class I.
Conclusions:
- SCAR system prevents unwanted immune recognition of genetically engineered cells.
- SCAR facilitates CRISPR-based studies of tumor-immune interactions.
- The SCAR system has significant implications for the clinical application of CRISPR technologies.
