Editing CCR5 in CD4+ T cells with high specificity
In order to assess the genotoxic potential of TALEN mediated editing ofCCR5 , we investigated the occurrence of off-target (OT) activity by two different means: targeted amplicon sequencing of in silicopredicted OTs or an unbiased in cellula assay. The top 20 potential OTs were predicted using the PROGNOS tool [28]. Amplicons from edited samples were compared to amplicons of untreated samples (Fig. 4A). Two of the top 20 OTs revealed some low but significant OT activity over background (Table S2). However, both OT01 (0.12% vs. 0.10% background, p =0.000211) and OT10 (0.08% vs. 0.07% background, p =0.004422) are situated in intronic regions. These data were complemented with an unbiased OT detection approach termed oligonucleotide capturing assay (OCA) which is based on GUIDE-seq [36]. The highest score (OCA1) was obtained for the CCR5target site (Fig. 4B). While OT01 was also found by OCA (OCA03), OT10 did not match with any of top 24 OCA hits (Table S3). While CCR2did not come up as a potential OT in PROGNOS, OCA picked up a weak activity at CCR2 (OCA4). Importantly however, all identified OCA sites had a considerably lower score than the CCR5 on target site (OCA1). Together, these OT analyses demonstrate that the employed TALENs are highly specific designer nucleases with minimal OT activity.