Fig. 2: Robust and durable ablation of CCR5 surface expression in primary human T cells.

A–F Human PBMCs from 3 donors were stimulated with phytohemagglutinin (PHA) for 3 days, electroporated with either a “mock” non-specific gRNA targeting GFP (green), single gRNAs targeting CCR5 (TB7-pink, TB8-brown, TB48-blue, TB50-red), or a dual gRNA approach comprising TB48 + TB50 (purple). Each symbol represents a distinct donor. A 48-h post electroporation, gene editing was measured by Sanger sequencing of CCR5 amplified from genomic DNA. B FACS plots depict CCR5 surface expression on mock edited (GFP gRNA) or CCR5 edited CD4⁺ (left) and CD8⁺ (right) T cells in a representative donor 48-h post electroporation. CCR5 expression levels 48-h after electroporation with various gRNAs on (C) CD4⁺ and (D) CD8⁺ T cells. E Longitudinal expression of CCR5 in vitro on CD4⁺ (top) and CD8⁺ (bottom) T cells up to 10 days post electroporation. Data from 3 independent PBMC donors are averaged, and error bars show ± SD. Area-under-the-curve (AUC) analysis of total CCR5 expression on (F) CD4⁺ and (G) CD8⁺ T cells throughout 10 days in culture. Each data point represents the average of 3 technical replicates, and each data point represents a distinct biological replicate using different donor PBMCs. Error bars denote minimum to maximum values. *denotes p < 0.05 and statistics generated from the Mann–Whitney test and p values are one-tailed.