Journal: Nucleic Acids Research

Article Title: Direct delivery of Cas-embedded cytosine base editors as ribonucleoprotein complexes for efficient and accurate editing of clinically relevant targets

doi: 10.1093/nar/gkae1217

Figure Lengend Snippet: Highly efficient C-to-T editing in HSPCs by CE_16_eA3A. Human CD34 + HSPCs from one healthy donor were electroporated with 30 μM of CE_16_eA3A RNPs. The treated HSPCs were infused into NBSGW mice and BM was harvested 16 weeks after transplantation. ( A ) Base editing in unfractionated BM after 16 weeks as compared with input HSPCs. The input is one cell product and is divided to multiple mice for xenotransplant. ( B ) Comparison of human chimerism in mice receiving unedited or edited HSPCs. Human cells in BM from all mice were detected by flow cytometry using an anti-human CD45 antibody and anti-mouse CD45 antibody, the percentage of human cells was calculated as human CD45 + cells/(human CD45 + cells + mouse CD45 + cells) x 100. Each dot indicates one mouse recipient. ( C ) HbF induction quantified by HPLC in human erythroid cells from unedited HSPCs (mock) and engrafted BM. ( D ) Comparison of the percentage of fetal cells (F-cells) between the mock and edited groups. F-cells indicate HbF+ cells. The percentage of fetal cells in engrafted erythroid cells was measured by flow cytometry using an anti-human HbF antibody. Data are plotted as mean ± SD ( n = 4 mice receiving edited cells and n = 3 mice receiving unedited cells).

Article Snippet: For flow cytometry analysis, the BM was blocked with Human TruStain FcXTM (BioLegend, #422302) and TruStain fcXTM (anti-mouse CD16/32; Biolegend, #101320) for 15 min at room temperature, followed by a 30-min incubation on ice with Fixable Viability Dye (Thermo Fisher Scientific, # 65-0865-18), mCD45 monoclonal antibody (Thermo Fisher Scientific, #61-0451-82), Mouse Anti-Human CD45 (BD Bioscience, #560367), anti-human CD235a (BioLegend, # 349104), anti-human CD19 (BioLegend, #302212), anti-human CD33 (BioLegend, #366608), anti-human CD34 (BioLegend, #343504) and anti-human CD3 (BioLegend, #300420).

Techniques: Transplantation Assay, Comparison, Flow Cytometry