Targeted Gene Correction Minimally Impacts Whole-Genome Mutational Load in Human-Disease-Specific Induced Pluripotent Stem Cell Clones

• Published in: Cell stem cell Vol. 15; no. 1; pp. 31 - 36
• Main Authors: Suzuki, Keiichiro, Yu, Chang, Qu, Jing, Li, Mo, Yao, Xiaotian, Yuan, Tingting, Goebl, April, Tang, Senwei, Ren, Ruotong, Aizawa, Emi, Zhang, Fan, Xu, Xiuling, Soligalla, Rupa Devi, Chen, Feng, Kim, Jessica, Kim, Na Young, Liao, Hsin-Kai, Benner, Chris, Esteban, Concepcion Rodriguez, Jin, Yabin, Liu, Guang-Hui, Li, Yingrui, Izpisua Belmonte, Juan Carlos
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.07.2014
• Subjects: Adenoviridae - genetics; Clone Cells; CRISPR-Cas Systems - genetics; DNA Repair - genetics; Endonucleases - genetics; Endonucleases - metabolism; Genetic Therapy; Genetic Vectors - genetics; Genetic Vectors - metabolism; Genome - genetics; HEK293 Cells; Humans; Induced Pluripotent Stem Cells - physiology; Mutation - genetics; Regenerative Medicine; Sequence Analysis, DNA
• ISSN: 1934-5909; 1875-9777; 1875-9777
• DOI: 10.1016/j.stem.2014.06.016

The utility of genome editing technologies for disease modeling and developing cellular therapies has been extensively documented, but the impact of these technologies on mutational load at the whole-genome level remains unclear. We performed whole-genome sequencing to evaluate the mutational load at single-base resolution in individual gene-corrected human induced pluripotent stem cell (hiPSC) clones in three different disease models. In single-cell clones, gene correction by helper-dependent adenoviral vector (HDAdV) or Transcription Activator-Like Effector Nuclease (TALEN) exhibited few off-target effects and a low level of sequence variation, comparable to that accumulated in routine hiPSC culture. The sequence variants were randomly distributed and unique to individual clones. We also combined both technologies and developed a TALEN-HDAdV hybrid vector, which significantly increased gene-correction efficiency in hiPSCs. Therefore, with careful monitoring via whole-genome sequencing it is possible to apply genome editing to human pluripotent cells with minimal impact on genomic mutational load. [Display omitted] •Mutational load in gene-corrected diseased iPSC clones was assessed by WGS.•Gene-corrected single-cell clones show low incidences of sequence variation.•TALEN-mediated gene-corrected clones show little evidence of off-target effects.•A TALEN-HDAdV hybrid vector shows high genome-editing efficiency in hiPSCs. Whole-genome sequencing of disease-specific human iPSCs corrected by helper-dependent adenoviral vectors or TALENs shows low mutational load and few off-target effects.