Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells

Susan M. Byrne, Luis Ortiz, Prashant Mali, John Aach, George M. Church
Source: Nucleic Acids Res
Publication Date: (2015)
Issue: 43(3): e21
Research Area:
Cancer Research/Cell Biology
Basic Research
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
4D-Nucleofector™ X-Unit
Human iPSC transfection All plasmids were purified using the Qiagen Endo-free Plasmid Maxiprep kit. Plasmids were nucleofected into iPSC cells using the Lonza 4D-Nucleofector X unit (Buffer P3, Program CB-150) according to manufacturer\\\\\\\'s instructions. For each 20 µl nucleofection reaction, 0.2–0.5 × 106 iPSC were transfected with up to 4 µg of plasmid DNA. Post-nucleofection, iPSC were plated onto 24- and 96-well Matrigel-coated plates containing mTesr1 media plus 10 µM Y-27632. For CRISPR-based nucleofections with a targeting vector (Figures ?(Figures11 and ?and2,2, and Supplementary Figures S2–S7), 2 µg of targeting vector plasmid, 0.5 µg of Cas9 plasmid, and 1.5 µg of total sgRNA plasmid were used. When two sgRNAs were used, 0.75 µg of each plasmid was combined. When no sgRNAs were used, 1.5 µg of pUC19 was used instead. For CRISPR-based nucleofections without a targeting vector (Figure ?(Figure33 and Supplementary Figures S10 and S11), 2 µg of total plasmid was used: 0.5 µg of Cas9 plasmid with 0.75 µg of each sgRNA plasmid or pUC19. For TALEN-based nucleofections with a targeting vector (Supplementary Figure S5), 2 µg of targeting vector plasmid plus 2 µg total of TALEN plasmid was used. For one dsDNA break using one TALEN pair, 1 µg of each TALEN-expressing heterodimer plasmid was used. For two dsDNA breaks using two TALEN pairs, 0.5 µg of each TALEN-expressing heterodimer plasmid was used.
Sequence-specific nucleases such as TALEN and the CRISPR/Cas9 system have so far been used to disrupt, correct or insert transgenes at precise locations in mammalian genomes. We demonstrate efficient 'knock-in' targeted replacement of multi-kilobase genes in human induced pluripotent stem cells (iPSC). Using a model system replacing endogenous human genes with their mouse counterpart, we performed a comprehensive study of targeting vector design parameters for homologous recombination. A 2.7 kilobase (kb) homozygous gene replacement was achieved in up to 11% of iPSC without selection. The optimal homology arm length was around 2 kb, with homology length being especially critical on the arm not adjacent to the cut site. Homologous sequence inside the cut sites was detrimental to targeting efficiency, consistent with a synthesis-dependent strand annealing (SDSA) mechanism. Using two nuclease sites, we observed a high degree of gene excisions and inversions, which sometimes occurred more frequently than indel mutations. While homozygous deletions of 86 kb were achieved with up to 8% frequency, deletion frequencies were not solely a function of nuclease activity and deletion size. Our results analyzing the optimal parameters for targeting vector design will inform future gene targeting efforts involving multi-kilobase gene segments, particularly in human iPSC.