Targeted nicks stimulate gene addition by homology-directed repair (HDR) without inducing mutations characteristic of nonhomologous end joining (NHEJ). (A) Illustration of experimental procedure. Cells were transfected or transduced with the indicated ZFN variants in the presence of a RFLP-containing donor DNA. Genomic DNA was collected 3–10 d post-transfection or -transduction and subjected to a RFLP assay to confirm DNA repair via HDR in panel B or a Surveyor nuclease assay to detect the induction of indels characteristic of repair via NHEJ in panel C. The integration of a patch sequence introduces an additional BglI site to the CCR5 locus. Upon BglI digestion, 86-bp, 926-bp, and 1553-bp products will be generated from the patch integrated allele, whereas an 86-bp and 2519-bp product will be generated from the wild-type allele. Note that the 86-bp product is not visible (it has been run off the gel). For the Surveyor nuclease assay, two additional products (estimated sizes are ∼66 bp and 97 bp, respectively) will be generated by the induction of indels caused by NHEJ repair at the ZFN target site. The uncleaved “parental” PCR product is 163 bp. (B) K562 cells were transfected with the indicated ZFN combinations in the presence of a CCR5-patch donor (Supplemental Fig. S2A). Genomic DNA was collected 10 d post-transfection and subjected to a RFLP assay. The percentage of BglI-sensitive DNA (indicated by arrows) resulting from targeted integration of the patch sequence is indicated below each lane. (C) The samples in panel B were also analyzed for the presence of indels using the Surveyor nuclease assay. (Arrows) Specific cleavage products. Numbers at the bottom of each lane indicate the percentage of CCR5 alleles containing indels, i.e., % of Surveyor nuclease cleaved products (indicated by arrows). (D) Nick-induced targeted gene addition in primary human cells. Primary human fibroblast cells were transduced with a lenti-CCR5-patch vector (30 MOI) in the absence (No ZFN) or presence of indicated Ad5/F35 ZFN vectors (300 MOI). Cells were collected 3 d post-transduction, genomic DNA isolated, and a RFLP assay performed. The percentage of BglI-sensitive DNA (indicated by arrows) resulting from targeted integration of the patch sequence is indicated below each lane.
