Abstract
Studying the cis-regulatory elements (CREs) of genes in Th17 cells during autoimmune disease progression, such as experimental autoimmune encephalomyelitis (EAE), is often limited by the availability of gene-edited mice. Here, we present a protocol for CRISPR-mediated deletion of a CRE in murine Th17 cells for in vivo assessment of effector function in EAE. We describe steps for dual U6gRNA construction, preparation of retroviruses, viral delivery, and Th17 differentiation. We then detail procedures for in vivo functionality analysis. For complete details on the use and execution of this protocol, please refer to Zhong et al.1,2.