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Efficient, footprint-free human iPSC genome editing by consolidation of Cas9/CRISPR and piggyBac technologies.

Nat Protoc. 2017; 
Wang Gang,Yang Luhan,Grishin Dennis,Rios Xavier,Ye Lillian Y,Hu Yong,Li Kai,Zhang Donghui,Church George M,Pu Willi
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Recombinant Proteins mM EDTA) NaCl (Boston BioProducts, cat. no. BM-244) 10× Taq PCR Buffer (GenScript, cat. no. B0005 Get A Quote

摘要

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 gu... More

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