Deletion of the highly conserved functional region of the Cd4 enhancer NCE in RLM11 cells using CRISPR/Cas9 reduces CD4 expression

Abstract

Abstract The glycoprotein cell surface marker CD4 is a key mediator in the adaptive immune response and T-cell development. Signaled CD4+ CD8+ double positive cells (DP) upregulate CD4 on the cell surface, allowing for a persistent TCR signal in the thymus and differentiating into the CD4+ single positive helper lineage. Previously, we have demonstrated that NCE (novel cis element), located in the first intron of Cd4, acts as an enhancer in RLM11 cells (CD4+, CD8−, Th-Pok− thymoma, arrested at the intermediate stage of development), but not in AKR-1G1 or VL3-3M2 cells (DP thymomas). We have identified a highly conserved region within NCE, labeled core-NCE, which may be vital to the enhancer’s function. To investigate the contribution of core-NCE to the level of CD4 expression, we utilized CRISPR/Cas9 technology to target the region for deletion. Here we describe the construction of plasmids containing guide sequences for targeted deletion of core-NCE by Cas9 cutting and NHEJ repair. We have successfully generated and sequence verified plasmids with three upstream and three downstream guides and introduced them in RLM11 cells. After single cell sorting, we obtained one clone that has the 300bp core-NCE deleted on one Cd4 allele, which resulted in 66% reduction in CD4 expression from that allele. As we removed the core-NCE in cells that have already established CD4 expression, we conclude that the core NCE has an enhancer function at the intermediate/transitional stage of thymocyte development that is separate from the previously described epigenetic function of the same intronic region, making NCE a good candidate for the regulatory element responsible for CD4 upregulation during differentiation into the CD4 lineage.