Efficient, footprint-free human iPSC genome editing by consolidation of Cas9/CRISPR and piggyBac technologies
- 8 December 2016
- journal article
- research article
- Published by Springer Nature in Nature Protocols
- Vol. 12 (1), 88-103
- https://doi.org/10.1038/nprot.2016.152
Abstract
This protocol uses a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon for robust, highly efficient and scarless genome editing of human iPSCs, enabling a parental line to be engineered to harbor many separate mutations. Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker-free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of ∼1–5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with guide RNA (gRNA), donor DNA and piggyBac transposase resulted in efficient, targeted genome editing and concurrent scarless transgene excision. Using this approach, in 7 weeks it is possible to efficiently obtain genome-edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40–50% and homology-directed repair (HDR) frequencies of 10–20%.Keywords
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