27
Life Technologies
™
| lifetechnologies.com/parkinsons
Figure 13. Generation of PD donor iPSCs with LRRK2 G2019S corrected to wild type.
(A) Sequence of LRRK2 G2019S region in the PD line. The binding sites for the TAL pair are
underlined in red. The TALs were electroporated into the cells along with a 1 kb purified
PCR fragment containing the wild type sequence and 500 bp flanking sequences. (B)
Colony screening by GeneArt
®
Genomic Cleavage Detection assay. Out of the 140 colonies
screened, colonies 26 and 27 showed negligible cleavage product due to mismatch,
indicating that the heterozygous mutation was mainly corrected to homozygous wild type.
(C) TaqMan
®
SNP Genotyping Assay confirmed that clones 26 and 27 and their daughter
colonies contain homozygous wild type allele, as these clones (red) plotted in the same
region as the wild type controls (from WT donor plasmid or WT template from HEK 293
cells) on the allelic discrimination plot. (D) Ion PGM
™
sequencing results showed progress
from the heterozygous state of the parental line (53% G), to a predominantly edited form
in colony 26 (93% G), and finally to a homozygous edited state (100% G) in each of three
daughter colonies.
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Allelic discrimination plot
Homozygous Mutant – control
Homozygous Wild Type – control
Parental – Heterozygous Mutant
Edited daughter
colonies
confirmed
Edited colonies identified
No Template – negative control
Legend
Homozygous Allele 1/Allele 1
Heterozygous Allele 1/Allele 2
Allele
2
Allele 1
Homozygous Allele 2/Allele 2
Undetermined
Sample sequenced Wild type (G) Mutant (A)
Parental—heterozygous 53 47
LRRK2-edited colony 26 93 7
Edited daughter colony 1 100 0
Edited daughter colony 2 100 0
Edited daughter colony 3 100 0
C
D
Results
