Genome Editing in Human Stem Cells

Susan M. Byrne, Prashant Mali, and George M. Church
Source: Methods Enzymol
Publication Date: (2014)
Issue: 546: 119-37
Cells used in publication:
Embryonic Stem Cell (ES), human
Species: human
Tissue Origin: embryo
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
4D-Nucleofector® X-Unit

optimized protocol for ES cells, P3 and CB-150


The use of custom-engineered sequence-specific nucleases (including CRISPR/Cas9, ZFN, and TALEN) allows genetic changes in human cells to be easily made with much greater efficiency and precision than before. Engineered double-stranded DNA breaks can efficiently disrupt genes, or, with the right donor vector, engineer point mutations and gene insertions. However, a number of design considerations should be taken into account to ensure maximum gene targeting efficiency and specificity. This is especially true when engineering human embryonic stem (ES) or induced pluripotent stem cells (iPSCs), which are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, we describe a protocol for easily engineering genetic changes in human iPSCs, through which we typically achieve targeting efficiencies between 1–10% without any subsequent selection steps. Since this protocol only uses the simple transient transfection of plasmids and/or single-stranded oligonucleotides, most labs will easily be able to perform it. We also describe strategies for identifying, cloning, and genotyping successfully edited cells, and how to design the optimal sgRNA target sites and donor vectors. Finally, we discuss alternative methods for gene editing including viral delivery vectors, Cas9 nickases, and orthogonal Cas9 systems.