Solexa-Illumina deep sequencing of ZFN-treated cells.

(A) Short 31bp reads detect ZFN-induced non-homologous end-joining (NHEJ) events in HEK293T cells. The FokI cutting region (

CAACTA) is indicated in bold, deletions are shown with “−” and insertions are underlined. (B) 104bp-read protocol: quantifying the rate of insertions and deletions induced by ZFN under a PGK promoter. SF268, K562 and BT549 cells were kept at 37°C or subjected to transient cold shock to increase NHEJ (30°C; [63]). ZFN plasmid amounts (0, 1 and 1.5 µg) are indicated by −, + asnd ++, respectively. Obligate heterodimer FokI mutants [57] are indicated where used (ObH). (C) Controls using wt∶mutant plasmids at ratios of 100∶1 and 1000∶1. After mixing, samples were treated identically as for other Solexa samples (3bp-barcode PCR, adapter ligation etc.). The proportion of wt∶mutant sequence in the Solexa output was then calculated. (D) Quantifying the rate of insertion of a barcoded (wt coding sequence) donor plasmid into the genomic p53 locus, in SF268 cells. CMV and PGK promoters were tested in combination with wt or ObH FokI nuclease domains. (E) A similar gene repair experiment in BT549 cells. Genomic DNA was collected 7 days after ZFN and donor plasmid transfection, to reduce background.