Schematic of the assay outlined by Noori et al. The assay begins with the identification of germline variants affecting PRF1, UNC13D, STX11, or STXBP2, the genes associated with fHLH, in patients diagnosed with or suspected of having HLH. To study the functional effects of these variants in vitro, especially VUS, mouse CD8 T cells are isolated and CRISPR/Cas9 genome editing is used to knock out the mouse gene corresponding to the human gene of interest (ie, the gene harboring a variant). Knockout of the endogenous mouse genes impairs the ability of gene-edited cells to upregulate CD107a expression (a marker of degranulation) and induce target cell killing. An expression vector is generated containing the relevant human cDNA, which is engineered to contain the variant identified in the patient (the DNA variant is depicted by a green star). After transducing the edited mouse CD8 T cells with this vector, expression of the human protein is confirmed by immunoblotting. Cells are then subjected to a CD107a (LAMP-1) expression assay to measure degranulation and a chromium release assay to measure their capacity to induce cytotoxicity. Expression of pathogenic fHLH gene variants fails to rescue degranulation and/or cytotoxicity, whereas expression of variants with residual function partially or fully rescues these functions. Figure prepared by Briana Williams, St. Jude Children’s Research Hospital.