Life Technologies
™
| Parkinson's cell models 16
TAL-based editing has been reported to have fewer
off-target cleavage events than observed with other
technologies. We sought to test for such possible off-target
events in the cell lines derived from the MSA donor.
To assess the possibility of off-target modifications having
arisen in the edited iPSC lines, exome sequencing was
performed to compare the exomes of the two edited cell
lines to the parental line (Figure 6). To generate the exome
sequencing data, the Ion AmpliSeq
™
Exome Kit was used in
conjunction with the Ion Proton
™
Sequencer and a custom
Ion Reporter
™
Software analysis workflow was designed
to identify indels, which are the most likely results of
undesired off-target cleavage.
Exome sequencing for off-target modifications
Ion AmpliSeq™ Exome Kit
Ion Proton™ Sequencer
Ion Reporter™ Software
Ion Proton
™
Sequencer Ion AmpliSeq
™
Exome Kit Ion Reporter
™
Software
Figure 6. Workflow for screening for off-target cleavage events. Ion AmpliSeq
™
exome libraries were prepared for parental and edited lines using the Ion AmpliSeq
™
Exome Kit and
sequenced with the Ion Proton
™
Sequencer. Sequence data were uploaded to and analyzed by Ion Reporter
™
Cloud Software. A custom Ion Reporter
™
workflow was designed and applied,
based on a variation of a standard tumor versus normal (germline) workflow. Any putative off-target variants were then analyzed by Sanger sequencing.
Methods
