Targeted gene correction minimally impacts whole-genome mutational load in human-disease-specific induced pluripotent stem cell clones

Abstract

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.

Fig. 1. Relationship between gene-corrected clones and their parental lines

(A) Parental disease iPSC lines were genetically corrected by HDAdV or TALEN. During the gene-correction process, neomycin resistant colonies were isolated and gene-correction events were determined by genotyping. Identified gene-corrected clones were expanded and genomic DNA was extracted for WGS at day 80. The neomycin-resistance cassette was removed by FLPo recombinase, and Neo-removed clones were expanded and genomic DNA was extracted for WGS at day 150 to 180. The results of WGS analysis were compared between each parental line and their genetically modified clone to determine mutations accumulated during the entire process. (Group 1) HGPS disease iPSC line (HGPS) and their gene-corrected clone by HDAdV (cHGPS) were published previously (Liu et al., 2011a; Liu et al., 2011b). (Group 2) SCD disease iPSC line (SCD) and their gene-corrected clone by HDAdV (cSCD) were published previously (Li et al., 2011). (Group 3) PD disease iPSC line (PD) and their gene-corrected clone by HDAdV (cPD) were published previously (Liu et al., 2012). (Group 4) The single cell derived SCD disease iPS (SCD-ref) was genetically corrected by HDAdV or TALEN. HDAdV1 and TALEN1 target intron 1 of HBB gene. HDAdV2 and TALEN2 target downstream of HBB gene. The unmodified clone (SCD-ctrl) was used as a strict control. (B) Gene-targeting and gene-correction efficiencies at the HBB locus with TALEN, HDAdV, CRISPR-CAS nuclease and the TALEN-HDAdV combination vector (telHDAdV). See also Figure S1.