Extended Data Fig. 3: Related to main Fig. 2.
From: In vivo site-specific engineering to reprogram T cells
a, Cells were treated with VSVG-WT or αCD3-EDVs carrying Cas9 and a sgRNA targeting CLTA, and AAV6 or AAV-hT7 carrying a HDR template to knock-in a reporter sfGFP transcript in exon 1 of CLTA. GFP expression is only observed if correctly integrated in CLTA. Cells were treated with 3 × 105 sgRNA/cell EDV, and with 5 × 105 vg/cell AAV. GFP expression was analysed by flow cytometry at least 72 h after treatment and genomic integration was confirmed by dPCR. Primary human T cells (n = 3), NK cells (n = 2), CD34+ HSC (n = 3), Macrophages (n = 3). Results are the mean ± SEM from two or three donors (n = 2 or 3). b, Four cell lines from B cell malignancies NALM6, Raji, SupB15 JeKo were treated according to d. GFP expression was analysed by flow cytometry at least 72 h after treatment. c, Human CD34+ hematopoietic stem cells were electroporated with Cas9/sgCLTA and treated with AAV6 or AAV-hT7 carrying a HDR template to knock-in a reporter sfGFP transcript in exon 1 of CLTA. Cells were treated 5 × 104 vg/cell AAV. GFP expression was analysed by flow cytometry at least 72 h after treatment (left panel) and genomic integration was confirmed by dPCR (right panel). Results are the mean ± SEM from three donors (n = 3). Significance was assessed using a two-tailed unpaired t-test. d, dPCR analysis on genomic DNA extracted from samples in (d) to confirm genomic integration at CLTA. Including knock-in analysis of treated NALM6-ffLuc-GFP cells treated the same way as d (flow cytometry analysis not available due to ubiquitous GFP expression). Data presented as percent of edited alleles. Results are the mean ± SEM from three donors (n = 3).
