Abstract
Genome engineering technology using engineered nucleases has been rapidly developing, enabling the efficient correction of simple mutations. However, the precise correction of structural variations (SVs) such as large inversions remains limited. Here we describe a detailed procedure for the modeling or correction of large chromosomal rearrangements and short nucleotide repeat expansions using engineered nucleases in human induced pluripotent stem cells (hiPSCs) from a healthy donor and patients with SVs. This protocol includes the delivery of engineered nucleases with no donor template to hiPSCs, and genotyping and derivation/characterization of gene-manipulated hiPSC clones. With engineered nucleases, genomic inversions, reversions, and deletions of short nucleotide expansions can be identified in 2 weeks, and desired clones can be generated in as little as 3–4 weeks. This protocol enables the correction of large inverted segments and short nucleotide repeat expansions in diseases such as hemophilia A, fragile X syndrome, Hunter syndrome, and Friedreich's ataxia.
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Acknowledgements
We thank D.-S. Jang from the Medical Research Support Section, Yonsei University College of Medicine, for assistance with graphics. D.-W.K. was supported by grants from the National Research Foundation of Korea (the Bio and Medical Technology Development Program, grant nos. 2012M3A9B4028631 and 2012M3A9C7050126) and from the Korean Ministry of Health and Welfare (grant no. HI15C0916). I.-H.P. was supported in part by the NIH (grant nos. GM0099130-01 and GM111667-01) and the CSCRF (grant nos. 12-SCB-YALE-11 and 13-SCB-YALE-06).
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C.-Y.P. conceived and developed the protocol. C.-Y.P. and J.J.S. optimized the protocol, performed the experiments, and wrote the manuscript. S.-H.C. and D.R.L. helped with optimization of the protocol and manuscript preparation. I.-H.P. and D.-W.K. supervised the experiments and wrote the manuscript.
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Park, CY., Sung, J., Choi, SH. et al. Modeling and correction of structural variations in patient-derived iPSCs using CRISPR/Cas9. Nat Protoc 11, 2154–2169 (2016). https://doi.org/10.1038/nprot.2016.129
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DOI: https://doi.org/10.1038/nprot.2016.129
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