Systematic CRISPR-mediated functional fine mapping of inborn errors of immunity causal genes in primary human T cells and hematopoietic stem cells: To predict all IL2RG mutations that cause X-SCID 4748

Description The genetic causes for many inborn errors of immunity (IEI) patients remain elusive, partly due to the inability to distinguish pathogenic mutations from the abundant, mostly benign variants of uncertain significance (VUS). We developed a novel CRISPR-Cas9 knock-in platform in primary cells to assess the functional impact of single nucleotide variants (SNVs) in a defined genomic region. We applied this platform to map SNVs across exon 5 of IL2RG, the causal gene of X-linked severe combined immunodeficiency (X-SCID). SNVs were recreated in T cells in a pooled format, and cell fitness was evaluated under IL-2 selection pressure. We interrogated the function of all SNVs and determined that all known pathogenic (ClinVar; HGMD) X-SCID SNVs imparted decreased T cell fitness while benign SNVs had no impact on T cell fitness. Of 16 ClinVar VUS, 13 were predicted damaging and 2 benign. We identified 71 unclassified missense SNVs as potentially pathogenic. Select screen-predicted loss-of-function (LOF) SNVs were validated by individual SNV knock-ins into HSCs, which were differentiated into lymphocytes in vitro. These LOF SNVs blocked T-cell differentiation while permitting B-cell differentiation, recapitulating the X-SCID lymphocyte profile. We identified novel LOF SNVs in IL2RG exon 5 that could lead to X-SCID but have yet to be found in patients. The results warrant further investigation of the remaining IL2RG exons and establish a methodology to study other disease-causing genes. Funding Sources Supported by NIH P01; NIH K08; NIH T32; Hearts to Humanity Eternal Research Grant. Topic Categories Translational and Interventional Immunology (TI)