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Summary: Generating knockout mice for target molecules in specific T cell populations, without subset-specific promoters, is time-consuming and costly. Here, we describe steps for enriching mucosal-associated invariant T cells from the thymus, expanding them in vitro and performing a CRISPR-Cas9 knockout. We then detail procedure for injecting the knockout cells into wounded Cd3ε−/− mice and characterizing them in the skin.For complete details on the use and execution of this protocol, please refer to du Halgouet et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.