Fig. 3

The TGFβ RII locus is successfully targeted via CRISPR/Cas9 system. A Schematic illustration of genomically knocking out TGFβ RII taking advantage of synthetic gRNAs targeting the TGFβRII locus and IVT-RNA of the nuclease Cas9, followed by IVT-RNA based CAR expression in human T-cells. B Graphic representation of the sequence and genomic location of three different gRNAs targeting exon2 of TGFβ RII and 20 bases in length, arrows indicate the polarity of the gRNAs. C T7 endonuclease 1 assay showing extra bands for gRNA treated T-cell groups in contrast to the control group, confirming indel formation in the gRNA treated group. D Histogram sequencing results for wild type and gRNA7 treated groups, elicit a heterologous sequence 4 bases inside the gRNA target target sequence upstream of complementary 5’-NGG PAM-sequence, which is in line with indel formation. E Bar graph of TIDE analysis illustrates that all three different gRNAs can lead to successful TGFβ RII knockout in both human CD4+ and CD8+ T-cells. In all experiments, mean ± SD of three technical replicates are given and experiments, involving T cells, are repeated for at least three donors