Isolation of single-base genome-edited human iPS cells without antibiotic selection

Abstract

Precise editing of human genomes in pluripotent stem cells by homology-driven repair of targeted nuclease-induced cleavage has been hindered by the difficulty of isolating rare clones. We developed an efficient method to capture rare mutational events, enabling isolation of mutant lines with single-base substitutions without antibiotic selection. This method facilitates efficient induction or reversion of mutations associated with human disease in isogenic human induced pluripotent stem cells.

Figure 1. Using ddPCR to detect a point mutation in human iPS cells

(a) Our mutagenesis and mutation detection strategy for PHOX2B is shown. We designed a pair of TALENs that target the mutation site and a 60-nt oligonucleotide DNA donor with the C→A substitution in the middle. The position of a common primer pair and allele-specific probes conjugated with different fluorophores are indicated. (b) ddPCR with plasmid mixtures. Mixtures containing the indicated concentrations of the mutant allele in a wild-type background were analyzed. Yellow lines indicate borders between different samples. Red dots represent droplets containing the mutant allele. (c) ddPCR with genomic DNA from iPS cells harboring the mutant allele induced by the PHOX2B TALENs. The mutagenized iPS cells were plated in a 96-well plate and 8 wells were analyzed for the mutant and wild-type alleles. An unrelated TALEN pair targeting the PRKAG2 locus was used as a negative control (Neg). (d) Sequencing results of isolated clones after PHOX2B mutagenesis.

Figure 2. Point mutagenesis in human iPS cells

(a) Overview of the approach to isolate rare mutants. Cells transfected with a TALEN pair and a donor oligonucleotide harboring the desired point mutation are immediately plated into a 96-well plate. The plates are replicated so that one 96-well plate is cryopreserved, while the other is used for DNA analysis. The mutant allelic frequency is measured from pooled cells from several wells by ddPCR. The well with the highest mutant frequency identifies the well in the cryopreserved plate that is used for re-plating into another 96-well plate for sib-selection. This process can be repeated until the mutants are sufficiently enriched for clonal isolation. iPS cell clones are isolated from the pool with the greatest enrichment of mutated cells and genotyped. (b) Enrichment of PRKAG2 mutant cells by sib-selection. The mutant allelic frequency as measured by ddPCR after the first, second, and third sib-selections is shown. On the left are shown the raw droplet data for the negative controls, the best well, and three random examples. Unrelated PHOX2B TALENs were used as negative controls for the first sib-selection, whereas completely negative “sib” wells are shown as the negative controls for the second and third sib-selections. The calculated mutant allelic frequency from the best wells is shown on the right. The average frequency after the first sib-selection is also shown. (c) Sequencing results for a representative isolated heterozygous clone from the sib-selection screen for the PRKAG2 mutant and a wild-type control.