Functional Interrogation of Primary Human T Cells via CRISPR Genetic Editing

Abstract

Developing precise and efficient gene editing approaches using CRISPR in primary human T cell subsets would provide an effective tool in decoding their functions. Toward this goal, we used lentiviral CRISPR/Cas9 systems to transduce primary human T cells to stably express the Cas9 gene and guide RNAs that targeted either coding or noncoding regions of genes of interest. We showed that multiple genes (<i>CD4</i>, <i>CD45</i>, <i>CD95</i>) could be simultaneously and stably deleted in naive, memory, effector, or regulatory T cell (Treg) subsets at very high efficiency. Additionally, nuclease-deficient Cas9, associated with a transcriptional activator or repressor, can downregulate or increase expression of genes in T cells. For example, expression of glycoprotein A repetitions predominant (GARP), a gene that is normally and exclusively expressed on activated Tregs, could be induced on non-Treg effector T cells by nuclease-deficient Cas9 fused to transcriptional activators. Further analysis determined that this approach could be used in mapping promoter sequences involved in gene transcription. Through this CRISPR/Cas9-mediated genetic editing we also demonstrated the feasibility of human T cell functional analysis in several examples: 1) <i>CD95</i> deletion inhibited T cell apoptosis upon reactivation; 2) deletion of <i>ORAI1</i>, a Ca²⁺ release-activated channel, abolished Ca²⁺ influx and cytokine secretion, mimicking natural genetic mutations in immune-deficient patients; and 3) transcriptional activation of <i>CD25</i> or <i>CD127</i> expression enhanced cytokine signaling by IL-2 or IL-7, respectively. Taken together, application of the CRISPR toolbox to human T cell subsets has important implications for decoding the mechanisms of their functional outputs.