Protocol to establish endogenous knockins in Jurkat T-ALL cells using CRISPR-Cas9-mediated homology-directed repair.

Jurkat cells are a versatile cell-line model utilized across multiple areas of biology. Here, we present a protocol for inserting DNA sequences (knockins) at an endogenous locus using CRISPR-Cas9 technology via homology-directed repair (HDR). We describe steps for designing, constructing, and validating endogenous knockins in T-ALL Jurkat cells. This protocol has potential application in gene regulatory studies, cancer biology, HIV research, and T-cell signaling, among others. For complete details on the use and execution of this protocol, please refer to Costa JR et al.1.