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

Authors:
Wang G, Yang L, Grishin D, Rios X, Ye LY, Hu Y, Li K, Zhang D, Church GM, Pu WT.
In:
Source: Nat Protocols
Publication Date: (2017)
Issue: 12(1): 88-103
Research Area:
Stem Cells
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Platform:
Nucleofector™ I/II/2b
Experiment
iPSC line: cells were detached by Versene, after stopping with mTESR and Y-27632 cells are spinned down ans resuspended in human stem cell solution 1 Add 1µSUPER piggyBac plasmid and 5µg pPB-rtTA-hCas9-puro-PB per 100µl sample. transfer the mixture to the cuvette and nucleofct via program B-016. After adding 500µl mTESR (with ROCK inhibitor) transfer the cells to matrigel coated wells.
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 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%.