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Modeling and correction of structural variations in patient-derived iPSCs using CRISPR/Cas9

Nature Protocols volume 11, pages 2154–2169 (2016)Cite this article

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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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Figure 1: NHEJ-dependent genome engineering strategies.
Figure 2: HDR-dependent genome engineering strategies.
Figure 3: Targeting strategies and PCR-based screening.
Figure 4: Harvest of colonies for genotype screening.
Figure 5: Corrected expression of F8 and FMR1.

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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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  1. Chul-Yong Park and Jin Jea Sung: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physiology, Yonsei University College of Medicine, Seoul, Korea

    Chul-Yong Park, Jin Jea Sung, Sang-Hwi Choi, Dongjin R Lee & Dong-Wook Kim

  2. Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea

    Chul-Yong Park, Jin Jea Sung, Sang-Hwi Choi, Dongjin R Lee & Dong-Wook Kim

  3. Department of Genetics, Yale Stem Cell Center, Yale School of Medicine, New Haven, Connecticut, USA

    Chul-Yong Park & In-Hyun Park

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Contributions

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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Correspondence to In-Hyun Park or Dong-Wook Kim.

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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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