6080
ATGCTGCCATCATTGCAAAGATTGCTGACTACRGCATTGCTCAGTACTGCTGTAGAATGG
500 bp…GCTGACTACGGCATTGCTCAGTACT…500 bp
(~1 kb donor fragment)
ATGCTGCCATCATTGCAAAGATTGCTGACTACGGCATTGCTCAGTACTGCTGTAGAATGG
ATGCTGCCATCATTGCAAAGATTGCTGACTACAGCATTGCTCAGTACTGCTGTAGAATGG
6030
Forward LRRK2 TAL Reverse LRRK2 TAL
6040 6050 6060 6070
26 Life Technologies
™
| Parkinson's cell models
To establish cell models to study the role of LRRK2 G2019S
and GBA N370S mutations in PD-relevant phenotypes, we
generated GeneArt
®
Precision TAL–edited iPSC lines with
these mutations corrected to wild type. Correcting the
LRRK2 G2019S mutation back to wild type required editing
via homologous recombination, which involved changing
one nucleotide from an A back to the wild type G using a
GeneArt
®
Precision TALs pair flanking the region along with
a 1 kb donor DNA containing the desired correction (Figure
13A). In our initial screen for the LRRK2 correction, 2 out of
140 colonies (1.4%) were positive for editing in the GeneArt
®
Genomic Cleavage Detection assay (Figure 13B, colonies 26
and 27). The colonies were subcloned and rescreened with
the TaqMan
®
SNP Genotyping assay (Figure 13C). Ion PGM
™
sequencing was performed on positive colonies to confirm
clonality of the population.
Correction of the LRRK2 G2019S
mutation in the PD-3 line
A
B
