GSE137657
Generation of targeted homozygosity in the genome of human induced pluripotent stem cells
When loss of heterozygosity (LOH) is correlated with loss or gain of disease phenotype, it is often necessary to identify which gene or genes are involved. Here, we generated a region-specific LOH-inducing system based on mitotic crossover in human induced pluripotent stem cells (hiPSCs). We first tested our system on chromosome 19. To detect homozygous clones generated by LOH, a positive selection cassette was inserted at the AASV1 locus of chromosome 19. LOHs were generated by the combination of allele-specific double-stranded DNA breaks introduced by CRISPR/Cas9 and suppression of Bloom syndrome (BLM) gene expression by the Tet-Off system. The BLM protein inhibitor, ML216, exhibited a similar crossover efficiency and distribution of crossover sites. We next applied this system to the short arm of chromosome 6 where human leukocyte antigen (HLA) loci are located. Genotyping and fluorescence-activated cell sorting analysis demonstrated that LOHs associated with chromosomal crossover occurred at the expected positions. Thus, HLA-homozygous hiPSCs generated from parental cells may help to mitigate a current shortcoming of hiPSC-based transplantation related to the immunological differences between donor and host. In conclusion, the system described in this study would greatly facilitate not only the genetic analysis of hiPSCs but also hiPSCs-based  regenerative medicine. Overall design: RNA-sequencing analyses were performed in six fibroblast cells and one its-derived induced pluripotent stem cells.