Optimized protocol to generate genome-wide inactivated Cas9-expressing murine T cells

In vivo genome-wide CRISPR screens in primary T cells allow the systematic and unbiased identification of non-redundant regulatory mechanisms shaping immune responses. Here, we present an optimized protocol for efficient generation of a pool of genome-wide inactivated Cas9-expressing T cells using a retroviral library of sgRNA. We detail the process of large-scale viral production and library integration in activated murine T cells as well as the two-step PCR approach for sgRNA recovery and abundance evaluation. For complete details on the use and execution of this protocol, please refer to Sutra Del Galy et al. (2020).