title: Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system.
abstract: Precise gene correction using the CRISPR-Cas9 system in human iPS cells holds great promise for various applications, such as the study of gene functions, disease modeling, and gene therapy. In this review article, we summarize methods for effective editing of genomic sequences of iPS cells based on our experiences correcting dystrophin gene mutations with the CRISPR-Cas9 system. Designing specific sgRNAs as well as having efficient transfection methods and proper detection assays to assess genomic cleavage activities are critical for successful genome editing in iPS cells. In addition, because iPS cells are fragile by nature when dissociated into single cells, a step-by-step confirmation during the cell recovery process is recommended to obtain an adequate number of genome-edited iPS cell clones. We hope that the techniques described here will be useful for researchers from diverse backgrounds who would like to perform genome editing in iPS cells.
authors.affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
authors.forename: Hongmei Lisa
authors.initials: HL
authors.lastname: Li
authors.affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
authors.forename: Peter
authors.initials: P
authors.lastname: Gee
authors.affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan; Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Ishida
authors.affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan; Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto, Japan. Electronic address: akitsu.hotta@cira.kyoto-u.ac.jp.
authors.forename: Akitsu
authors.initials: A
authors.lastname: Hotta
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems; D018929:Cell Culture Techniques; D000072669:Gene Editing; D015894:Genome, Human; D057809:HEK293 Cells; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000502:physiology*; D014162:Transfection
keywords: CRISPR; Gene correction; Genome editing; Knock-in; Knock-out; iPS cells