Faculty Sponsor: Sophia Sarafova
T cells are essential effectors in the body’s adaptive immune response with the CD4+ helper T cells serving as the coordinators. Error-free lineage commitment of developing T cells to the CD4+ helper lineage is highly dependent on the upregulation of CD4 expression as cells progress from the immature CD4+CD8+ precursor stage (DP) to the more mature CD4+ CD8low (INT) and CD4+ CD8- (CD4SP) stages. It has been known that the CD4 well characterized proximal enhancer (PE) is not responsible for upregulation of CD4 and that an additional enhancer must exist in the Cd4 locus that controls CD4 expression at the INT and CD4SP stages of development when the commitment to the CD4 lineage occurs. The novel cis-regulatory element (NCE) has been identified as a late-stage CD4 enhancer by transient transfections in RLM11 intermediate-stage thymoma cell lines, but not in cell lines arrested at the double positive stage when the proximal enhancer PE works the best. In order to verify that the NCE exhibits stage-specific activity in more natural circumstances than transient transfections, previous lab members used CRISPR/Cas9 mediated deletion of the most highly conserved segment of the NCE enhancer (Frag5) in RLM11 cells arrested at the INT stage and found that in the absence of one copy of NCE CD4 expression decreased by 25%. I used the same approach to delete the same fragment in the AKR1G1cells arrested at the DP stage of development, where NCE is not expected to function. To delete Frag 5, AKR1G1 cell cultures were transfected with two plasmids containing GFP, Cas9, and guide RNA sequences designed to target the two ends of Frag5. After 24 hours, individual cells green cells expressing GFP were sorted into 96-well plates. 150 CRISPRed clones were maintained and screened by PCR for successful deletions of Frag 5 on at least one allele and confirmed through sequencing. Many clones had a single allele deletion and at least one clone has a double deletion. Furthermore, CD4 expression in the CRISPRed clones was measured by immunostaining for CD4 and flow-cytometry and compared to the parental AKR1G1 cell line. In contrast to the transient transfection results, removing NCE in the natural context of the Cd4 locus affects levels of CD4 expression in cells arrested at the DP stage.