FIG. 5.

CoDA-syn nuclease activity at the cellular IDUA locus. (A and B) Nuclease 170 target site (A) before and (B) after nuclease cleavage resulting in staggered 5′, 4-bp overhangs. (C) Artificial oligonucleotide duplex (OD) with overhang regions compatible with a specific ZFN cleavage product and also containing phosphorothioate linkages (*) to prevent intracellular nuclease degradation. Detection of nuclease-dependent endogenous target site modification. The OD insertion event can be detected by using OD and genomic DNA-specific primers and characterized by DNA sequencing. Sequence analysis of genome editing. (D) OD insertion into the target site by perfect ligation. (E) OD target site insertion after DNA recession and repair by nonhomologous end joining resulting in a 15-bp insertion and a 95-bp deletion (i15/del95) followed by genomic sequence.