Improving homology-directed repair efficiency in human stem cells
Summary
The generation of induced pluripotent stem cell models of human disease requires efficient modification of one or both alleles depending on dominant or recessive inheritance of the disease. To faithfully recapitulate many disease variants, the introduction of a single base change is required. The introduction of additional silent mutations designed to prevent re-cutting of the modified allele by Cas9 is not an optimal strategy, particularly for non-coding variants. Here, we developed an improved protocol for efficient engineering of single nucleotide variants in human iPS cells. Using a fluorescent BFP->GFP assay to monitor the incorporation of a single base pair change, we optimized the protocol to achieve HDR in 70% of unselected human iPS cells. The additive effects of cold shock, a small molecule enhancer of HDR and chemically modified ssODN dramatically shift the bias of repair in favor of HDR, resulting in a seven-fold higher ratio of HDR to NHEJ from 0.5 to 3.7. Copyright © 2019 Elsevier Inc. All rights reserved.
| Authors | Skarnes WC, Pellegrino E, McDonough JA |
|---|---|
| Journal | Methods (San Diego, Calif.) |
| Publication Date | 2019 Jul 15;164-165:18-28 |
| PubMed | 31216442 |
| DOI | 10.1016/j.ymeth.2019.06.016 |