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Seamless genome editing in human pluripotent stem cells using custom endonuclease–based gene targeting and the piggyBac transposon

Nature Protocols volume 8, pages 2061–2078 (2013)Cite this article

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Abstract

I report here a detailed protocol for seamless genome editing using the piggyBac transposon in human pluripotent stem cells (hPSCs). Recent advances in custom endonucleases have enabled us to routinely perform genome editing in hPSCs. Conventional approaches use the Cre/loxP system that leaves behind residual sequences in the targeted genome. I used the piggyBac transposon to seamlessly remove a drug selection cassette and demonstrated safe genetic correction of a mutation causing α-1 antitrypsin deficiency in patient-derived hPSCs. An alternative approach to using the piggyBac transposon to correct mutations involves using single-stranded oligonucleotides, which is a faster process to complete. However, this experimental procedure is rather complicated and it may be hard to achieve homozygous modifications. In contrast, using the piggyBac transposon with drug selection–based enrichment of genetic modifications, as described here, is simple and can yield multiple correctly targeted clones, including homozygotes. Although two rounds of genetic manipulation are required to achieve homozygote modifications, the entire process takes ∼3 months to complete.

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Figure 1: DSB repair mechanisms.
Figure 2: The piggyBac transposon–mediated seamless modification in comparison with the Cre/loxP-based method.
Figure 3: Overview of the PROCEDURE for targeted modification using the piggyBac transposon system.
Figure 4: The targeting strategy.
Figure 5: The picking of hPSC colonies for lysate-PCR and subculture.
Figure 6: PCR-based screening of targeted clones.
Figure 7: PCR-based screening of transposon-excised clones.

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Acknowledgements

I thank E.P. Tan for careful reading of the manuscript, and S.T. Rashid and members of the Allan Bradley laboratory (Sanger Institute, UK) and the Ludovic Vallier laboratory (University of Cambridge, UK) for discussion and for sharing their experience. This work was supported by Wellcome Trust (WT077187).

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    Kosuke Yusa

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K.Y. developed the protocol and wrote the paper.

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Correspondence to Kosuke Yusa.

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Yusa, K. Seamless genome editing in human pluripotent stem cells using custom endonuclease–based gene targeting and the piggyBac transposon. Nat Protoc 8, 2061–2078 (2013). https://doi.org/10.1038/nprot.2013.126

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