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Generation of Targeted Homozygosity in the Genome of Human Induced Pluripotent Stem Cells

Generation of Targeted Homozygosity in the Genome of Human Induced Pluripotent Stem Cells

Yasuhide Yoshimura, Ayako Yamanishi, Tomo Kamitani, Jin-Soo Kim, Junji Takeda

PLoS One. 2019 Dec 5;14(12):e0225740.

PMID: 31805151

Abstract:

When loss of heterozygosity (LOH) is correlated with loss or gain of a disease phenotype, it is often necessary to identify which gene or genes are involved. Here, we developed 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 flow cytometric analysis demonstrated that LOHs associated with chromosomal crossover occurred at the expected positions. Although careful examination of HLA-homozygous hiPSCs generated from parental cells is needed for cancer predisposition and effectiveness of differentiation, they may help to mitigate the current shortcoming of hiPSC-based transplantation related to the immunological differences between the donor and host.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR4244075	ML216			Price

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