500 bp…GTCCTTACCCTAGAACCTCCTGTACCATGTGGTCGGCTGGACCGA…500 bp
Reverse GBA TAL Forward GBA TAL
(1 kb donor PCR fragment)
GBA
GBAP1
GCGCCTTT GTCT CTTT GCCTTT GTCCTT ACCCTA GAA CCT CCT GT ACCAT GT GGT CGGCT GGA CCGA CT GGA A CCTT GCCCT GAA CCCCGAA GGA GGA CCCAA TT GGGT GCGT AA CT T T GT CGA CA GT CCCAT CAT T GT A GA CA T CA
GCGCCTTT GTCT CTTT GCCTTT GTCCTT ACCCTA GAA CCT CCT GT ACCAT GT GGT CGGCT GGA CCGA CT GGA A CCTT GCCCT GAA CCCCGAA GGA GGA CCCAA TT GGGT GCGT AA CT T T GT CGA CA GT CCCAT CAT T GT A GA CA T CA
GAGCCTTT GTCT CTTT GCCTTT GT CCTT ACCCT A GAA CCT CCT GT ACCA T GT GGT CGGCT GGA CCGA CT GGA A CC-----------------------------------------------------------------------------------------------------------------CAT CAT T GT A GA CA T CA
9020 9030 9040 9050 9060 9070 9080 9090 9100 9110 9120 9130 9140 9150 9160
28 Life Technologies
™
| Parkinson's cell models
TAL-enabled correction of the GBA N370S mutation back
to wild type also used HDR. This work was complicated
by the presence of a pseudogene, GBAP1, which shares
complete homology with GBA in the N370S region. To avoid
the pseudogene, we designed a TAL pair that binds to a
nonhomologous region downstream of the N370S target
region (Figure 14A), and designed PCR primers for areas
of nonhomology with the pseudogene to generate clean
amplicons for screening. In our initial screen by TaqMan
®
SNP Genotyping Assay, 1 of 48 colonies (2%) was positive
for editing with a large shift in the population towards
wild type (Figure 14B). The colonies were subcloned and a
clonal population was isolated. Ion PGM
™
sequencing was
performed on positive colonies to confirm clonality of the
G-to-A correction (Figure 14C).
Similar to the MSA lines, both edited PD donor iPSC lines
were characterized to be karyotypically normal and able to
express all pluripotency markers (by immunocytochemistry
and TaqMan
®
hPSC Scorecard
™
Panel, data not shown).
A
Figure 14. Generation of PD donor iPSCs with GBA N370S corrected to wild type. (A) Sequence of GBA N370S (top) and pseudogene GBAP1 (bottom) 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 TaqMan
®
SNP Genotyping Assay. Out of 48 colonies screened (green) in the first round, one colony (colony 10) seemed to move towards the homozygous wild type
control on the allelic discrimination plot. After subcloning this colony, more colonies plotted in the same region as wild type control (from WT donor plasmid) on the allelic discrimination
plot of the second round screening. (C) Ion PGM
™
sequencing results showing the percent of mutations detected in the heterozygous parental line, colony 10 from the first round of
screening, a daughter colony of colony 10, and a later passage of the daughter colony. Ion PGM
™
sequencing was repeated for several daughter colonies. Clone 10–12 showed close to 100%
wild type nucleotide T at this location, indicating a clonal population.
Correction of the GBA N370S mutation
in the PD-3 line
