Fig. 6

Genomic characterization of CRISPR knock-in CAR-T cells. a On-target copy number estimation using ddPCR. Genomic DNA from scale up experiments (n = 3 independent donors) was analyzed in technical duplicates using primers/probe to target Albumin (reference gene, copy number = 2) and primers/probe to target the left insertion site of the GD2-CAR into TRAC. Copy number values were normalized to the frequency of CAR + cells as determined via flow cytometry. b Source of predicted off-target sites. c-e, Quantification of indels in predicted off-target sites and TRAC using CRISPAltRations for samples obtained from Donor 3 of large-scale experiments. Editing was binarily classified using a thresholded Fishers Exact test (p < 0.05) with limitations (> 0.5% indels in treatment; < 0.4% indels in control; > 5,000 reads) for edited samples with (c) knock-out, (d) knock-in without enrichment and (e) knock-in after enrichment (red circle = significant; blue circle = not significant). Indel frequencies were plotted against non-electroporated Mock control samples to highlight pre-existing indels and noise. Quadrants display the limits of classification (bottom left – treatment % indels < 0.5; top right – control % indels > 0.4%; top left – all limitations met and classifiable; bottom right – no limitations met). The top left quadrant contains classifiable events that occur in edited samples and indicates only on-target editing in these samples. f + g Representative insertion site analysis for Donor 3 samples of non-enriched (f) and enriched (g) GD2 knock-in CAR-T cells using TLA. GD2 CAR sequences were inserted into the TRAC locus on chromosome 14 without evidence for off-target insertion