title: Crispr-mediated Gene Targeting of Human Induced Pluripotent Stem Cells.
abstract: 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 or induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, we describe an optimized protocol for genome engineering of human iPSCs using a simple transient transfection of plasmids and/or single-stranded oligonucleotides. With this protocol, we achieve transfection efficiencies greater than 60%, with gene disruption efficiencies from 1-25% and gene insertion/replacement efficiencies from 0.5-10% without any further selection or enrichment steps. We also describe how to design and assess optimal sgRNA target sites and donor targeting vectors; cloning individual iPSC by single cell FACS sorting, and genotyping successfully edited cells.
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Byrne
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts.
authors.forename: George M
authors.initials: GM
authors.lastname: Church
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D018929:Cell Culture Techniques; D002469:Cell Separation; D002470:Cell Survival; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D018920:Coculture Techniques; D004720:Endonucleases / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D018390:Gene Targeting / Q000379:methods*; D005838:Genotype; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D051379:Mice; D010957:Plasmids / Q000378:metabolism; D017393:RNA Editing; D014162:Transfection
chemical_list: D004720:Endonucleases
keywords: CRISPR / Cas9 nuclease; gene targeting; genome engineering; human induced pluripotent stem cells; transfection