A 312 bp region surrounding the gol/slc24a5 Pair 14/15 cleavage site was amplified by PCR with primers 5′-ATCTGATATGGCCATGTCCAACATCG-3′ and 5′-GGAACAATCCCATACGCTCCTGCAG-3′ and a 226 bp region surrounding the ntl/Brachyury Pair 2/3 cleavage site with primers 5′-ACGAATGTTTCCCGTGCTCAGAGCC-3′ and 5′-GCTGAAAGATACGGGTGCTTTCATCCAGTGCG-3′. To analyze ZFN-induced mutations eliminating the BsrDI restriction site that lies between the left and right ZFN binding sites, PCR products were digested with BsrDI and resolved on a 2% agarose gel. BsrDI resistant sequences were 226 bp, while wild-type sequences were digested to 176 and 50 bp fragments. Heterozygous individuals carried all 3 band sizes. PCR products were ligated into the pCR® 4-TOPO® vector for sequencing (Invitrogen, Carlsbad CA).
For analysis of potential off-target action by the ZFNs, the position weight matrix (i.e., an experimentally determined consensus DNA binding site) for each ZFN was determined using an in vitro site selection method (Supplementary Information online ). The off-target sites, and all the genotyping data, are provided in Supplementary Fig. 11 online .
For analysis of potential off-target action by the ZFNs, the position weight matrix (i.e., an experimentally determined consensus DNA binding site) for each ZFN was determined using an in vitro site selection method (
