Figure 7 |. FACS balancing of on-target and off-target mutagenesis.
Fifty thousand CHO cells transfected with GFP-2A-ZFNLeft and RFP-2A-ZFNRight targeting B4GALT4 were bulk isolated for high or medium GFP and RFP double-fluorescence levels and analyzed by IDAA for indels at the on-target site in B4GALT4 and an off-target site in B4GALT3. Note that off-target mutagenesis occurs at the high nuclease level but not at the medium nuclease level, where on-target mutagenesis remains high. The size and frequency of selected indels are indicated. The “shoulder” indicated by an asterisk is not an indel, since it is also present in the control sample; it is likely due to incomplete 3´ A nucleotide addition to the IDAA amplicon (see Table 3, Step 21). The sequences of the on- and off-target sites are shown, with nuclease-binding sequence in uppercase and mismatches in boldface. IDAA was performed in an ABI 3130 instrument.