Fig 2: Genome editing in primary T cells — genotypic analysis.a Schematic of gene targeting strategy. Integration of a CAR–RQR8 construct is targeted to exon 2 of the CD52 locus. Gene targeting by homology-direct repair is mediated by a CRISPR-Cas9 nuclease in combination with an AAV6 donor vector that harbors the CAR–RQR8 expression cassette flanked by ˜500 bp long homology arms. EFS, elongation factor 1a short promoter; CD19-CAR, CD19-targeting CAR; P2A, 2A self-cleaving peptide of porcine teschovirus; RQR8, see schematic; polyA, poly-adenylation signal. b Genotyping by T7E1 assay. The percentage of cleaved products is indicated. c Genotyping by NGS analysis. Pie charts show fractions of alleles with indels. d Genotyping by in–out junction PCR. Primer binding sites for 5' and 3'-junction PCRs as well as expected product lengths are indicated in panel (a). Indel, insertion/deletion mutation; UT, untreated T cells; AAV, AAV-transduced T cells; RNP, RNP-electroporated T cells; RNP + AAV, electroporated with RNP and transduced with 1 × 104, 3 × 104, and 5 × 104 genome copies/cell from left to right, respectively.