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

ABSTRACT: 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 genom

SUBMITTER: Wang G 

PROVIDER: S-EPMC5352979 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature