Factor Induced Reprogramming and Zinc Finger Nuclease Aided Gene Targeting Cause Different Genome Instability in {beta}-thalassemia iPSCs [Cell Biology]

March 20th, 2015 by Ma, N., Shan, Y.-L., Liao, B.-J., Kong, G.-Y., Wang, C., Huang, K., Zhang, H., Cai, X.-J., Chen, S.-B., Pei, D.-Q., Chen, N.-S., Pan, G.-J.

Generation of personalized iPSCs followed by targeted genome editing provides an opportunity for developing customized effective cellular therapies for genetic disorders. However, whether edited iPSCs harbor unfavorable genomic variations needs to be critically ascertained before their clinical application. To examine the mutation status of the edited iPSC genome and trace the origin of possible mutations at different steps, we have generated virus-free iPSCs from amniotic cells carrying homozygous point mutations in β-hemoglobin gene (HBB) that cause severe β-thalassemia (Thal), corrected the mutations in both HBB alleles by ZFN-aided gene targeting, and obtained the final HBB gene corrected iPSCs by excising the exogenous drug resistant gene with Cre recombinase. Through comparative genomic hybridization (CGH) and whole-exome sequencing, we uncovered 7 copy number variations (CNVs), 5 small insertions/deletions (Indels) and 64 single nucleotide variations (SNVs) in β-Thal iPSCs before the gene targeting step, and found a single small CNV and 19 Indels and 340 SNVs in the final gene-corrected β-Thal iPSCs. Our data revealed substantial but different genomic variations occurred at factor induced somatic cell reprogramming and ZFN aided gene targeting step, suggesting that stringent genomic monitoring and selection is needed both at the time of iPSC derivation and after gene targeting.
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