Isolation of single-base genome-edited human ips cells without

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1 Nature Methods Isolation of single-base genome-edited human ips cells without antibiotic selection Yuichiro Miyaoka, Amanda H. Chan, Luke M. Judge, Jennie Yoo, Miller Huang, Trieu D. Nguyen, Paweena P. Lizarraga, Po-Lin So & Bruce R. Conklin Supplementary Information Supplementary Figure Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Figure 5 Supplementary Figure 6 Supplementary Figure 7 Supplementary Figure 8 Supplementary Figure 9 Supplementary Figure Supplementary Table Supplementary Table 2 Supplementary Table 3 Supplementary Table 4 Supplementary Table 5 Quantitative performance of genotyping PCR and ddpcr Temperature optimization of the TaqMan probe-primer set for PHOX2B mutagenesis PHOX2B and PKP2 mutagenesis in HEK293T cells and comparison of ddpcr to the Surveyor assay Application of CRISPR/Cas to detection of mutagenesis by ddpcr ddpcr for the PHOX2B mutagenesis in human ips cells Targeting design, sib-selection, and genotyping of the PRKAG2 mutagenesis Pluripotency marker expression and karyotype of PRKAG2- Het line BAG3 mutagenesis Genomic DNA sequences of the TALEN target sites in the isolated ips cell lines Advantages of new method compared to direct cloning of ips cells to establish scarless mutant ips cell lines Genes and mutations engineered in this study Comparison of direct cloning, the piggybac-based system, and our new method for isolation of one mutant ips cell line On- and off-target sites for the PHOX2B and PRKAG2 TALENs predicted by TALENoffer (as a separated excel file) TALENs used in this study TaqMan primer-probe sets and oligonucleotide donor DNA used in this study Nature Methods: doi:.38/nmeth.284

2 Supplementary Figure Quantitative performance of genotyping PCR and ddpcr. a PHOX2B Locus E E2 E3 Left TALEN...CTGAGCCATCCAGAACCTTTTCAATGTATAAAATGGAATATTCTTACCTCAATTCCTCTGCCTACGAGTCCTGTATGGCTGGGATGGACACCTCGAGCCT......GACTCGGTAGGTCTTGGAAAAGTTACATATTTTACCTTATAAGAATGGAGTTAAGGAGACGGATGCTCAGGACATACCGACCCTACCTGTGGAGCTCGGA... Right TALEN PHOX2B mutant oligonucleotide donor GAATATTCTTACCTCAATTCCTCTGCCTAAGAGTCCTGTATGGCTGGGATGGACACCTCG PHOX2B WT allele Forward primer FAM WT probe...ctgagccatccagaaccttttcaatgtataaaatggaatattcttacctcaattcctctgcctacgagtcctgtatggctgggatggacacctcgagcct......gactcggtaggtcttggaaaagttacatattttaccttataagaatggagttaaggagacggatgctcaggacataccgaccctacctgtggagctcgga... PHOX2B mutant allele Forward primer VIC probe Reverse primer...ctgagccatccagaaccttttcaatgtataaaatggaatattcttacctcaattcctctgcctaagagtcctgtatggctgggatggacacctcgagcct......gactcggtaggtcttggaaaagttacatattttaccttataagaatggagttaaggagacggattctcaggacataccgaccctacctgtggagctcgga... Reverse primer b d PHOX2B mutant (VIC) signal (VIC) WT (FAM) , 4, 2, 3, % No template 5% % PHOX2B WT (FAM) signal Plasmid mixtures (PHOX2B mutant allelic proportion) %.%.% % % 5% % WT 9, WT 6, % %.% c e PHOX2B mutant (VIC) sgnal / PHOX2B WT (FAM) signal.2 P = % %.%.% % %.%.% % %..% % %.% %.% % 5% % % Calculated PHOX2B mutant allelic frequency (%) % 4 2 % Nature Methods: doi:.38/nmeth.284

3 (a) Detailed TALEN targeting design using the TaqMan PCR system. PHOX2B mutagenesis is shown as an example. We designed a pair of TALENs that target the mutation site and a 6-nt oligonucleotide DNA donor with the Cà A substitution in the middle. A common primer pair and allele-specific probes conjugated with different fluorophores were also designed. At least one of the two primers was designed to anneal outside of the donor oligonucleotide sequence to amplify only on-target integrations. This figure is essentially identical to Fig. a, but the gene structure and sequence are shown here. (b) Genotyping PCR for the PHOX2B mutant allele. We used the plasmid mixtures (,.,,, 5, and % of the mutant allele) as templates. The wild-type and mutant alleles were detected by the FAM- or VIC-conjugated probe, respectively, by using a 384-well format real-time PCR machine. Triplicates of plasmid samples containing, 5, and % of the mutant allele in the wild-type allele background clustered together, whereas samples containing <% of the mutant templates clustered together. This assay is not highly sensitive, but is useful to genotype cloned cell lines. (c) Quantification of the genotyping PCR results with the plasmid mixtures. The ratio of the mutant (VIC) signal per the wild-type (FAM) signal is shown. The graphs for,., and % of the mutant allelic frequency are magnified on the left. These three samples are not significantly different (P =.2, one-way ANOVA). In contrast, the difference between and % samples was significant (P =.7, Student s t-test, upper right panel), indicating that the detection limit of genotyping PCR for a mutant allele is around %. The error bars are ± s.e.m. (n = 3). The log-scale plot of the data is shown below the linear plot. (d) ddpcr for the PHOX2B mutant and the wild-type alleles. Plasmid mixtures with different proportions (,.,.,,, 5, and % of the mutant allele) were analyzed. Red and blue dots represent droplets containing the mutant and the wild-type alleles, respectively. Note that the probes weakly cross-detected the wild-type and mutant alleles, which was not surprising since the two alleles differ by only a single nucleotide. However, this cross-hybridization can be excluded from quantitative analysis in ddpcr by setting the threshold of detection high enough to discriminate true-positive droplets from false-positive droplets, as shown here. The upper plot for the mutant allele is the same as in Fig. b, but shown here with the plot for the wild-type allele. (e) Quantification of the ddpcr results with the plasmid mixtures. The graphs for,.,., and % of the mutant allelic proportion are magnified in the middle, and those for and.% were further magnified on the left. ddpcr showed robust quantitative performance up to.%, since the difference between. and.% was statistically significant, but that between and.% was not. Error bars are ± s.e.m. (n = 3). P values calculated by Student s t-test (n = 3) are shown in the middle and left panels. The log-scale plot of the same data is also shown below the linear plot. Nature Methods: doi:.38/nmeth.284

4 Supplementary Figure 2 Temperature optimization of the TaqMan probe-primer set for PHOX2B mutagenesis. Extension and annealing temperature (C ) PHOX2B mutant (VIC) signal PHOX2B WT (FAM) signal 6, 4, 2, 2, 8, 4, WT WT After droplet generation, gradient PCR from 5 to 58 C was performed to find the optimal annealing and extension temperature for ddpcr. The data for the wild-type (FAM) and mutant (VIC) allele signal are shown. Red and blue dots represent droplets containing the mutant and wild-type alleles, respectively. At lower temperatures, the signal amplitudes were increased to give better separation between the negative and positive populations. Because the amplitudes were saturated around 5 C, this temperature was chosen for annealing and extension. We performed similar optimizations for the other probe-primer sets to find the optimal annealing and extension temperature (data not shown). Nature Methods: doi:.38/nmeth.284

5 Supplementary Figure 3 PHOX2B and PKP2 mutagenesis in HEK293T cells and comparison of ddpcr to the Surveyor assay. a e PHOX2B TALEN Negative Strong Weak PKP2 locus E6 E7 b NHEJ frequency (%) NegativeStrong Weak PHOX2B TALEN c (VIC) WT (FAM) 6, 4, 2, 2, 8, 4, PHOX2B TALEN Negative Strong Weak droplets WT droplets d allelic frequency (%) Negative E Strong Weak PHOX2B TALEN Left TALEN...TGCTTACGCTGACGGAGAATATCATCATCCCCTTTTCTGGGTGGCCTGAAGGAGACTACCCAAAAGCAAATGGTTTGCTCGATTTTGACATATTCTACAACGTCACTGGATGCC......ACGAATGCGACTGCCTCTTATAGTAGTAGGGGAAAAGACCCACCGGACTTCCTCTGATGGGTTTTCGTTTACCAAACGAGCTAAAACTGTATAAGATGTTGCAGTGACCTACGG... Right TALEN PKP2 mutant oligonucleotide donor GGAGAATATCATCATCCCCTTTTCTGGGTAGCCTGAAGGAGACTACCCAAAAGCAAATGG PKP2 WT allele Forward primer WT probe...tgcttacgctgacggagaatatcatcatccccttttctgggtggcctgaaggagactacccaaaagcaaatggtttgctcgattttgacatattctacaacgtcactggatgcc......acgaatgcgactgcctcttatagtagtaggggaaaagacccaccggacttcctctgatgggttttcgtttaccaaacgagctaaaactgtataagatgttgcagtgacctacgg... PKP2 mutant allele Forward primer VIC FAM probe Reverse primer...tgcttacgctgacggagaatatcatcatccccttttctgggtagcctgaaggagactacccaaaagcaaatggtttgctcgattttgacatattctacaacgtcactggatgcc......acgaatgcgactgcctcttatagtagtaggggaaaagacccatcggacttcctctgatgggttttcgtttaccaaacgagctaaaactgtataagatgttgcagtgacctacgg... Reverse primer f PKP2 TALEN Negative Strong Weak g NHEJ frequency (%) Negative Strong Weak PKP2 TALEN h (FAM) WT (VIC) 8, 6, 4, 2, 5, 4, 3, 2,, PKP2 TALEN Negative Strong Weak droplets WT droplets i allelic frequency (%) Negative Strong Weak PKP2 TALEN Nature Methods: doi:.38/nmeth.284

6 (a) Surveyor assay for the PHOX2B TALENs in HEK293T cells. We constructed two TALEN pairs that targeted the same DNA sequences but differed in activity (strong and weak, see Methods for details). Arrowheads indicate the digested DNA fragments due to NHEJ induced by the PHOX2B TALENs. The unrelated PRKAG2 TALEN pair was used as a negative control. (b) Quantification of the Surveyor assay. Error bars are ± s.e.m.; P values were calculated by Student s t-test (n = 3). (c) ddpcr for the PHOX2B mutagenesis in HEK293T cells with the strong and weak TALEN pairs. (d) Quantification of ddpcr. Error bars are ± s.e.m.; P values were calculated by Student s t-test (n = 3). ddpcr clearly detected the induced mutant allele, even with the weak TALEN pair. (e) PKP2 mutagenesis design. The design is similar to the PHOX2B mutagenesis, except a part of the forward primer and the entire reverse primer anneal outside of the donor oligonucleotide DNA. Also, specific probes for the wild-type allele and the mutant allele are conjugated with VIC and FAM, respectively. (f) Surveyor assay for PKP2 TALENs in HEK293T cells. The strong PHOX2B TALENs were used as a negative control. The two arrowheads indicate the digested DNA fragments due to NHEJ induced by the PKP2 TALENs. Similar to the results with the PHOX2B TALENs, the strong PKP2 pair robustly induced NHEJ in HEK293T cells. (g) Quantification of the Surveyor assay. Error bars are ± s.e.m.; P values were calculated by Student s t-test (n = 3). (h) ddpcr for the PKP2 mutagenesis in HEK293T cells. (i) Quantification of the ddpcr. Error bars are ± s.e.m.; P values were calculated by Student s t-test (n = 3). The P value between the control and the weak TALEN pair is much less significant for the Surveyor assay than for the ddpcr assay, indicating that ddpcr is the more sensitive and robust method. Nature Methods: doi:.38/nmeth.284

7 Supplementary Figure 4 Application of CRISPR/Cas to detection of mutagenesis by ddpcr. a RBM2 locus E8 RBM2 mutant oligonucleotide donor RBM2 WT allele Forward primer acagatatggcccagaaaggccgcggtctagtagtccggtgagccggtcactctccccga...ggaggtgtgaagattctaaatcctgctccttggctccctcacagatatggcccagaaaggccgcggtctcgtagtccggtgagccggtcactctccccgaggtcccacactcccagcttcacctcctgcagc......cctccacacttctaagatttaggacgaggaaccgagggagtgtctataccgggtctttccggcgccagagcatcaggccactcggccagtgagaggggctccagggtgtgagggtcgaagtggaggacgtcg... RBM2 mutant allele Forward primer E9 E Cas9 and grna GGTCTCGTAGTCCGGTGAGCCGG...ggaggtgtgaagattctaaatcctgctccttggctccctcacagATATGGCCCAGAAAGGCCGCGGTCTCGTAGTCCGGTGAGCCGGTCACTCTCCCCGAGGTCCCACACTCCCAGCTTCACCTCCTGCAGC......cctccacacttctaagatttaggacgaggaaccgagggagtgtcTATACCGGGTCTTTCCGGCGCCAGAGCATCAGGCCACTCGGCCAGTGAGAGGGGCTCCAGGGTGTGAGGGTCGAAGTGGAGGACGTCG... FAM VIC WT Reverse Primer...ggaggtgtgaagattctaaatcctgctccttggctccctcacagATATGGCCCAGAAAGGCCGCGGTCTAGTAGTCCGGTGAGCCGGTCACTCTCCCCGAGGTCCCACACTCCCAGCTTCACCTCCTGCAGC......cctccacacttctaagatttaggacgaggaaccgagggagtgtcTATACCGGGTCTTTCCGGCGCCAGATCATCAGGCCACTCGGCCAGTGAGAGGGGCTCCAGGGTGTGAGGGTCGAAGTGGAGGACGTCG... E Reverse Primer b allele WT allele c 6, 4, 2, 8, 6, 4, 2, allelic frequency (%).5 Negative grna Negative grna.28 RBM2 grna RBM2 grna (a) Design of the CRISPR/Cas system and the TaqMan PCR system for the RBM2 mutagenesis. We designed a grna that directs Cas9 to the mutation site to cleave this site, a 6-nt oligonucleotide donor that has the mutation in the middle, and an allelic-specific TaqMan PCR assay. (b) ddpcr for the RBM2 mutagenesis using CRISPR/Cas in HEK293T cells. A grna for the adeno-associated virus integration site (AAVS) was used as a negative control. (c) Quantification of ddpcr for the RBM2 mutagenesis by CRISPR/Cas. Only the RBM2 grna induced point mutagenesis. Error bars are ± s.e.m.; P-value was calculated by Student s t-test (n = 3). Nature Methods: doi:.38/nmeth.284

8 Supplementary Figure 5 ddpcr for the PHOX2B mutagenesis in human ips cells. a (VIC) 6, 4, 2, PHOX2B TALEN-mutagenized ips cell pool (well number) Neg WT b 3 2 PHOX2B mutant allelic frequency (%) WT (FAM) 2, 8, 4, WT Negative Well number c PHOX2B-mutant Het PHOX2B-mutant Homo X Y X Y (a) ddpcr for the PHOX2B mutant and wild-type alleles. We transfected ips cells with the strong PHOX2B TALENs and the oligonucleotide donor and plated the cells into a 96-well plate as cell pools. The mutant allelic frequency was measured for eight wells. The PRKAG2 TALENs were used as a negative control. The upper plot for the mutant allele is the same as in Fig. c, shown here together with the plot for the wild-type allele. (b) Quantification of ddpcr. The average of the PHOX2B mutant allelic frequency for the eight wells was 2.2%, whereas the best well, well 5, had 2.6%. We isolated PHOX2B- Het and Homo clones shown in Fig. d from well 5 and well 3, respectively. (c) Karyotype of the heterozygous and homozygous PHOX2B mutant ips cell lines. Both clones maintained a normal male karyotype. Each chromosome is annotated by chromosomal number, X, or Y. Nature Methods: doi:.38/nmeth.284

9 a Supplementary Figure 6 Targeting design, sib-selection, and genotyping of the PRKAG2 mutagenesis. PRKAG2 Locus E4 Left TALEN E5...tgttaattttcacgtgtcctgcttttctctttgcagGTCCATCGGCTGGTGGTGGTAAATGAAGCAGATAGTATTGTGGGTATTATTTCCCTGTCGGACATTCTGCAAGC......acaattaaaagtgcacaggacgaaaagagaaacgtcCAGGTAGCCGACCACCACCATTTACTTCGTCTATCATAACACCCATAATAAAGGGACAGCCTGTAAGACGTTCG... Right TALEN E6 b PRKAG2 mutant allelic frequency (%) PHOX2B 3 μg μg PRKAG2 PRKAG2 mutant oligonucleotide donor d f PRKAG2 WT allele Forward primer gtgtcctgcttttctctttgcaggtccatgggctggtggtggtaaatgaagcagatagta...tgttaattttcacgtgtcctgcttttctctttgcaggtccatcggctggtggtggtaaatgaagcagatagtattgtgggtattatttccctgtcggacattctgcaagc......acaattaaaagtgcacaggacgaaaagagaaacgtccaggtagccgaccaccaccatttacttcgtctatcataacacccataataaagggacagcctgtaagacgttcg... PRKAG2 mutant allele PRKAG2 mutant allelic frequency (%) PRKAG2 mutant (VIC) Forward primer Reverse primer...tgttaattttcacgtgtcctgcttttctctttgcaggtccatgggctggtggtggtaaatgaagcagatagtattgtgggtattatttccctgtcggacattctgcaagc......acaattaaaagtgcacaggacgaaaagagaaacgtccaggtacccgaccaccaccatttacttcgtctatcataacacccataataaagggacagcctgtaagacgttcg Reverse primer Clone 2.9 (Het) 5% FAM VIC WT probe probe PRKAG2 mutant (VIC) Well number st (3 clones) 2nd (22 clones) 3rd (65 clones) Clone 2.2 (Het) 5% PRKAG2 mutant (VIC) e c PRKAG2 mutant allelic frequency (%) PRKAG2 mutant allelic frequency (%) N Well number Het clones 5% Well number g. No template Wild-type PRKAG2 WT (FAM). No template Wild-type PRKAG2 WT (FAM). No template Wild-type PRKAG2 WT (FAM) PRKAG2 mutant (VIC) 6, 4, 2, droplets: 884 Allelic frequency: 49.97% PRKAG2 WT (FAM) 6, 4, 2, WT droplets: 885 Allelic frequency: 5.3% WT Nature Methods: doi:.38/nmeth.284

10 (a) Targeting design for PRKAG2 mutagenesis. A part of the forward primer and the entire reverse primer anneal outside of the donor oligonucleotide DNA. (b) ddpcr for the PRKAG2 mutant allele after transfection of different amounts of TALENs. We transfected ips cells with 3 or µg each of the PRKAG2 TALEN vector with the donor and plated the cells into a six-well plate. The PHOX2B TALENs were used as a negative control. The higher amount of the PRKAG2 TALENs induced more HR events. (c) ddpcr for the mutant allele after the first sib-selection. We transfected ips cells with the PRKAG2 TALENs and the donor, and plated the cells into a 96-well plate. The PHOX2B TALENs were used as a negative control (N). The allelic frequency was measured in 2 wells, and even the best well, well 2 (pink bar) had a mutant allelic frequency of only.4%. The cells in well 2 were split into another 96-well plate. (d) ddpcr for the mutant allele after the second sib-selection. The allelic frequency was measured in 39 wells and the best well, 2.27, had an approximate mutant allelic frequency of.73% (pink bar). Cells from well 2.27 were split into another 96-well plate. (e) ddpcr for the mutant allele after the third sib-selection. Of the 9 wells measured, the best well, , had an allelic frequency of about 6.8% (pink bar). Clones were isolated from well (f) Plot of genotyping PCR for the isolated clones. We genotyped a total of 8 ( ) clones in three separate reactions. Heterozygous clones are highlighted in green rectangles. We found clones were heterozygous for the PRKAG2 mutation. The data of sequence, expression of pluripotency marker genes, and karyotype for clone 2.2 and clone 2.9 are shown in Fig. 2c and Supplementary Fig. 7a and 7b as PRKAG2- Het and PRKAG2- Het 2, respectively. (g) ddpcr for the PRKAG2- Het clone. Purity of the isolated PRKAG2-Het (clone 2.2) was validated by ddpcr. The number of droplets positive for the mutated and wild-type alleles were 884 (49.97%) and 885 (5.3%), respectively, showing that the clone is a pure heterozygous line. These results demonstrate that our ddpcr method has an outstanding quantitative power. Nature Methods: doi:.38/nmeth.284

11 Supplementary Figure 7 Pluripotency marker expression and karyotype of PRKAG2-mutant Het line. a PRKAG2-mutant Het TRA--8 SOX2 Nuclei Merged PRKAG2-mutant Het 2 TRA--8 SOX2 Nuclei Merged SSEA4 OCT4 Nuclei Merged SSEA4 OCT4 Nuclei Merged b PRKAG2-mutant Het PRKAG2-mutant Het X Y X Y Independent PRKAG2-mutant Het X Y (a) Expression of TRA--8 (green), SOX2 (red), SSEA4 (green), and OCT4 (red) in isolated PRKAG2- Het and PRKAG2- Het 2 ips cell lines. Pluripotency markers were co-stained with DAPI to label all nuclei (blue). The merged images are also shown (Merged). The clones expressed all the pluripotency markers analyzed. Scale bars: μm. (b) Karyotype of the heterozygous PRKAG2 mutant ips cell lines. The PRKAG2- Het and PRKAG2- Het 2 lines were isolated from the same transfection of ips cells (clones 2.2 and 2.9 shown in Supplementary Fig. 6f, respectively). The Independent PRKAG2- Het line is another heterozygous PRKAG2 mutant line isolated from an independent transfection of ips cells by the new scarless method. All the clones maintained a normal male karyotype. Each chromosome is annotated by chromosomal number, X, or Y. Nature Methods: doi:.38/nmeth.284

12 Supplementary Figure 8 BAG3 mutagenesis. a BAG3 Locus E2 E3 E4 Left TALEN...GGCGGCAGCAGCGGCTCCTCAGAGGTCCCAGTCACCTCTGCGGGGCATGCCAGAAACCACTCAGCCAGATAAACAGTGTGGACAGGTGGCAGCGGCGGCGGCAGCCCAGCCCCCAGCCTCCCACGGACCTGAGgtaaggagaggccaggctcaccagcc......CCGCCGTCGTCGCCGAGGAGTCTCCAGGGTCAGTGGAGACGCCCCGTACGGTCTTTGGTGAGTCGGTCTATTTGTCACACCTGTCCACCGTCGCCGCCGCCGTCGGGTCGGGGGTCGGAGGGTGCCTGGACTCcattcctctccggtccgagtggtcgg... Right TALEN BAG3 mutant oligonucleotide donor TGCCAGAAACCACTCAGCCAGATAAACAGCGTGGACAGGTGGCAGCGGCGGCGGCAGCCC b BAG3 WT allele Forward primer WT Probe...GGCGGCAGCAGCGGCTCCTCAGAGGTCCCAGTCACCTCTGCGGGGCATGCCAGAAACCACTCAGCCAGATAAACAGTGTGGACAGGTGGCAGCGGCGGCGGCAGCCCAGCCCCCAGCCTCCCACGGACCTGAGgtaaggagaggccaggctcaccagcc......CCGCCGTCGTCGCCGAGGAGTCTCCAGGGTCAGTGGAGACGCCCCGTACGGTCTTTGGTGAGTCGGTCTATTTGTCACACCTGTCCACCGTCGCCGCCGCCGTCGGGTCGGGGGTCGGAGGGTGCCTGGACTCcattcctctccggtccgagtggtcgg... BAG3 mutant allele...ggcggcagcagcggctcctcagaggtcccagtcacctctgcggggcatgccagaaaccactcagccagataaacagcgtggacaggtggcagcggcggcggcagcccagcccccagcctcccacggacctgaggtaaggagaggccaggctcaccagcc......ccgccgtcgtcgccgaggagtctccagggtcagtggagacgccccgtacggtctttggtgagtcggtctatttgtcgcacctgtccaccgtcgccgccgccgtcgggtcgggggtcggagggtgcctggactccattcctctccggtccgagtggtcgg... BAG3 mutant allelic frequency (%) Forward primer c BAG3 WT VIC FAM Probe Reverse primer Reverse primer Mutated Left TALEN binding site Right TALEN binding site site CCAGTCACCTCTGCGGGGCATGCCAGAAACCACTCAGCCAGATAAACAGTGTGGACAGG. Before st 2nd BAG3-mutant Het Mutated Left TALEN binding site Right TALEN binding site site CCAGTCACCTCTGCGGGGCATGCCAGAAACCACTCAGCCAGATAAACAGT/CGTGGACAGG d TRA--8 Nuclei Merged SOX2 Nuclei Merged e SSEA4 Nuclei Merged OCT4 Nuclei Merged X Y Nature Methods: doi:.38/nmeth.284

13 (a) Targeting design for BAG3 mutagenesis. Both the forward and reverse primers anneal outside of the donor oligonucleotide DNA. The right TALEN recognizes the mutated site, but we were still able to conduct the mutagenesis. (b) Sib-selection for the BAG3 mutagenesis. The efficiency of BAG3 TALENs was similar to that of PRKAG2 TALENs, giving a.45% initial mutant allelic frequency. We successfully enriched the mutant cell population to 4.3% by two rounds of sib-selections. We then isolated a heterozygous mutant clone from this cell pool. (c) Genomic DNA sequences around the target site of the BAG3 TALEN pair in the BAG3-mutant Het line and the parental ips cell line, WTC (BAG3 WT). The left and right TALEN binding sites and the mutated site are highlighted. A DNA fragment, including this target site, was amplified by PCR with genomic DNA isolated from the parental ips cell lines as a template. The PCR amplicons were directly sequenced without cloning, which means the data are one-toone mixtures of paternal and maternal alleles. Therefore, if there were frame-shifts caused by NHEJ, sequencing peaks around the target site should have been frameshifted. However, both mutants showed clean single peaks identical to those in WTC, indicating that those clones have only HR events, but not NHEJ events, on the target site. (d) Expression of TRA--8, SOX2, SSEA4, and OCT4 (all green) in the isolated BAG3- Het ips cell line. Pluripotency markers were co-stained with DAPI to label all nuclei (blue). Merged images are also shown (Merged). The clone expressed all analyzed pluripotency markers. Scale bars: µm. (e) Karyotype of the BAG3- Het line. The clone maintained a normal male karyotype. Each chromosome is annotated by chromosomal number, X, or Y. Nature Methods: doi:.38/nmeth.284

14 Supplementary Figure 9 Genomic DNA sequences of the TALEN target sites in the isolated ips cell lines. a PHOX2B WT Left TALEN binding site Mutated site Right TALEN binding site TTCTTACCTCAATTCCTCTGCCTACGAGTCCTGTATGGCTGGGATGGACACCTCGAGCCT PHOX2B-mutant Homo PHOX2B-mutant Het Left TALEN binding site Mutated site Right TALEN binding site TTCTTACCTCAATTCCTCTGCCTAC/AGAGTCCTGTATGGCTGGGATGGACACCTCGAGCCT Left TALEN binding site Mutated site Right TALEN binding site TTCTTACCTCAATTCCTCTGCCTAAGAGTCCTGTATGGCTGGGATGGACACCTCGAGCCT b Left TALEN binding site Mutated site Right TALEN binding site CTTTTCTCTTTGCAGGTCCATCGGCTGGTGGTGGTAAATGAAGCAGATAGTATTGTGGGTATTAT PRKAG2-mutant Het PRKAG2-mutant Het 2 PRKAG2 WT Independent PRKAG2-mutant Het Left TALEN binding site Mutated site Right TALEN binding site CTTTTCTCTTTGCAGGTCCATC/GGGCTGGTGGTGGTAAATGAAGCAGATAGTATTGTGGGTATTAT Left TALEN binding site Mutated site Right TALEN binding site CTTTTCTCTTTGCAGGTCCATC/GGGCTGGTGGTGGTAAATGAAGCAGATAGTATTGTGGGTATTAT Left TALEN binding site Mutated site Right TALEN binding site CTTTTCTCTTTGCAGGTCCATC/GGGCTGGTGGTGGTAAATGAAGCAGATAGTATTGTGGGTATTAT Nature Methods: doi:.38/nmeth.284

15 (a) Genomic DNA sequences around the target site of the PHOX2B TALEN pair in the mutant ips cell lines (PHOX2B-mutant Het and PHOX2B-mutant Homo), as well as the parental ips cell line, WTC (PHOX2B WT). A portion of the same sequences just around the mutated sites are shown in Fig. d. The left and right TALEN binding sites and the mutated site are highlighted. A DNA fragment, including this target site, was amplified by PCR with genomic DNA isolated from these ips cell lines as a template. Similar to Supplementary Fig. 8c, the PCR amplicons were directly sequenced without cloning them. Both mutants showed clean single peaks identical to those in WTC, indicating that those clones have only HR events, but not NHEJ events, on the target site. (b) Genomic DNA sequences around the target site of the PRKAG2 TALEN pair in the mutant ips cell lines (PRKAG2- Het, PRKAG2- Het2, and Independent PRKAG2- Het), as well as WTC (PRKAG2 WT). A part of the same sequences just around the mutated sites of PRKAG2 WT and PRKAG2- Het are shown in Fig. 2c. The left and right TALEN binding sites and the mutated site are highlighted. The conditions for analysis were the same as for the PHOX2B mutant lines, and the PRKAG2 mutant ips lines also have only HR events, but not NHEJ events, on the target site. Nature Methods: doi:.38/nmeth.284

16 Supplementary Figure Advantages of new method compared to direct cloning of ips cells to establish scarless mutant ips cell lines. Mutagenesis New scarless method ddpcr sib-selection Cloning Direct cloning - Fewer clones to screen - Higher success rate - Higher time-efficiency - Labor intensive - Low success rate As the schematic shows, the use of direct cloning methods after mutagenesis, as is currently used, requires the isolation of hundreds of clones. Moreover, the mutagenesis frequency is not known before the isolation. Therefore, extensive screening is required to isolate and propagate a large number of clones, which can result in a complete failure to identify any positive mutants. In contrast, with new method, the frequency of mutated cells can be estimated before cloning. Furthermore, these mutated cells can be enriched by sib-selection so that only a small number of clones are required to obtain one mutant line. This approach greatly decreases hands-on time and labor while maximizing the chances of identifying desired clones. Nature Methods: doi:.38/nmeth.284

17 Supplementary Table Genes and mutations engineered in this study. Full Gene Name Nucleotide Amino Acid Reference PHOX2B Paired-like homeobox 2B 42C>A Tyr4Stop Trochet, D. et al. PKP2 Plakophilin-2 63G>A Trp538Stop Dalal, D. et al. 2 RBM2 RNA binding motif protein 2 96C>A Arg636Ser Brauch, K.M. et al. 3 PRKAG2 Protein kinase, AMP-activated, γ2 non-catalytic subunit 59C>G Arg53Gly Gollob, M.H. et al. 4 BAG3 BCL2-associated athanogene 3 45T>C Cys5Arg Villard, E et al. 5 Nature Methods: doi:.38/nmeth.284

18 Supplementary Table 2 Comparison of direct cloning, the piggybac-based system, and our new method for isolation of one mutant ips cell line. Task Direct Cloning piggybac System New Method Hands-on time for transfection of ips cells 2 hours 4 hours 2 hours Hands-on time for sib-selection None None 8 hours Clone number to isolate >2 96-well plate number required 6-2 <4 Hands-on time for feeding cells 5-25 hours 3 minutes hour Hands-on time for isolating clones > hours 3 minutes 3 minutes Hands-on time for genotyping of clones 6 hours 4 hours 3 minutes Total time required ~2.5 weeks ~ weeks ~8 weeks Total hands-on time required ~2-3 hours ~9 hours ~2 hours Restrictiveness in targeting design None Need a TTAA site None Factors for off-target damage Nucleases Nucleases Nucleases piggybac transposon Knowledge of correct targeting before cloning NO NO YES The time and effort to isolate one mutant ips cell line by direct cloning, the piggybac system, or our new method was estimated based on our results from mutagenesis in PRKAG2 and the reported piggybac system 6. The restrictiveness and fidelity of each mutagenesis strategy is also addressed. Note that with our new method, the researcher knows immediately after the first round of sib-selection whether to continue isolating clones or not, while with direct cloning or the piggybac system, the researcher proceeds to the end before knowing for sure that a mutant is isolated. Moreover, the piggybac system requires a TTAA site for piggybac insertion and has a risk to suffer from off-target damage by not only site-specific nucleases, but also the piggybac transposon. Nature Methods: doi:.38/nmeth.284

19 Supplementary Table 4 TALENs used in this study. PHOX2B Strong Pair PHOX2B Weak Pair PKP2 Strong Pair PKP2 Weak Pair PRKAG2 Pair BAG3 Pair Left TALEN Target DNA Sequence TACCTCAATTCCTCTGC (Addgene plasmid 49449) TACCTCAATTCCTCTGC (Addgene plasmid 49477) TGACGGAGAATATCATCA (Addgene plasmid 4945) TGACGGAGAATATCATCA (Addgene plasmid 49479) TCTCTTTGCAGGTCCAT (Addgene plasmid 49453) TCACCTCTGCGGGGCATG (Addgene plasmid 49465) Right TALEN Target DNA Sequence Left TALEN RVDs Right TALEN RVDs Spacer DNA Sequence TCGAGGTGTCCATCCCAGCC NG NI HD HD NG HD NI NI NG NG HD NN NI NN NN NG NN NG HD CTACGAGTCCTGTAT (Addgene plasmid 4945) NG HD HD NG HD NG NN HD HD NI NG HD HD HD NI NN HD HD TCGAGGTGTCCATCCCAGCC NG NI HD HD NG HD NI NI NG NG HD NK NI NK NK NG NK NG HD CTACGAGTCCTGTAT (Addgene plasmid 49478) NG HD HD NG HD NG NK HD HD NI NG HD HD HD NI NK HD HD TGGGTAGTCTCCTTCAGG NG NN NI HD NN NN NI NN NI NI NG NN NN NN NG NI NN NG HD NG TCCCCTTTTCTGGGTGG (Addgene plasmid 49452) NG NI NG HD NI NG HD NI HD HD NG NG HD NI NN NN TGGGTAGTCTCCTTCAGG NG NK NI HD NK NK NI NK NI NI NG NK NK NK NG NI NK NG HD NG TCCCCTTTTCTGGGTGG (Addgene plasmid 4948) NG NI NG HD NI NG HD NI HD HD NG NG HD NI NK NN TACCCACAATACTATCTGCT NG HD NG HD NG NG NG NN HD NG NI HD HD HD NI HD NI NI NG NI CGGCTGGTGGTGGTAAATGA (Addgene plasmid 49454) NI NN NN NG HD HD NI NG HD NG NI NG HD NG NN HD NG TCCACACTGTTTATCTG NG HD NI HD HD NG HD NG NN NG HD HD NI HD NI HD NG NN NG CCAGAAACCACTCAGC (Addgene plasmid 49466) HD NN NN NN NN HD NI NG NN NG NG NI NG HD NG NN The target DNA sequences and the RVDs of the TALENs used in this study are shown. Note that the first NG of the TALENs was included in the backbone plasmid. To create the weak TALENs, the NN RVDs in the strong TALENs (except the last NN of the PKP2 right TALEN) were substituted with the NK RVDs. The spacer DNA sequences of the wild-type alleles between the TALEN pairs and the Addgene plasmid numbers are also shown. The underlined nucleotides were mutated in this study. Nature Methods: doi:.38/nmeth.284

20 Supplementary Table 5 TaqMan primer-probe sets and oligonucleotide donor DNA used in this study. Forward Primer Reverse Primer Wild-Type Allele Specific Probe Allele Specific Probe PHOX2B CCATCCAGAACCTTTTCAATGTATAA GTGTCCATCCCAGCCATACAG AATTCCTCTGCCTACGA (FAM) CAATTCCTCTGCCTAAGA (VIC) PKP2 ACGCTGACGGAGAATATCATCA CCAGTGACGTTGTAGAATATGTCAAA CCCTTTTCTGGGTGGC (VIC) CTGGGTAGCCTGAA (FAM) RBM2 TGTGAAGATTCTAAATCCTGCTCCTT AGGAGGTGAAGCTGGGAGTGT CCGCGGTCTCGTAG (FAM) CGGTCTAGTAGTCC (VIC) PRKAG2 ATTTTCACGTGTCCTGCTTTTCTC CAGAATGTCCGACAGGGAAATAA AGGTCCATCGGCTG (FAM) AGGTCCATGGGCTG (VIC) BAG3 CAGCAGCGGCTCCTCAGA GTGAGCCTGGCCTCTCCTT CAGATAAACAGTGTGGACA (VIC) AGATAAACAGCGTGGACA (FAM) PHOX2B PKP2 RBM2 PRKAG2 BAG3 Oligonucleotide Donor DNA GAATATTCTTACCTCAATTCCTCTGCCTAAGAGTCCTGTATGGCTGGGATGGACACCTCG GGAGAATATCATCATCCCCTTTTCTGGGTAGCCTGAAGGAGACTACCCAAAAGCAAATGG ACAGATATGGCCCAGAAAGGCCGCGGTCTAGTAGTCCGGTGAGCCGGTCACTCTCCCCGA GTGTCCTGCTTTTCTCTTTGCAGGTCCATGGGCTGGTGGTGGTAAATGAAGCAGATAGTA TGCCAGAAACCACTCAGCCAGATAAACAGCGTGGACAGGTGGCAGCGGCGGCGGCAGCCC The primer and probe sequences for the TaqMan system used in this study are shown. The underlined nucleotides in the probes are specific to either the wild-type or the mutated alleles. The underlined nucleotides in the donors are introduced point mutations. Nature Methods: doi:.38/nmeth.284

21 References for Supplementary Infomation. Trochet, D. et al. Human mutation 3, E42-43 (29). 2. Dalal, D. et al. Circulation 3, (26). 3. Brauch, K.M. et al. Journal of the American College of Cardiology 54, (29). 4. Gollob, M.H. et al. Circulation 4, (2). 5. Villard, E. et al. European heart journal 32, (2). 6. Yusa, K. et al. Nature 478, (2). Nature Methods: doi:.38/nmeth.284