Susan M. Byrne, George M. Church
CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem cells and induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, an optimized protocol is described for genome engineering of human iPSCs using simple transient transfection of plasmids and/or single-stranded oligonucleotides without any further selection or enrichment steps. This protocol achieves transfection efficiencies >60%, with gene disruption efficiencies of 1-25% and gene insertion/replacement efficiencies of 0.5-10%. Details are also provided for designing optimal sgRNA target sites and donor targeting vectors, cloning individual iPSCs by single-cell FACS sorting, and genotyping successfully edited cells.
