Format

Send to

Choose Destination
Methods. 2016 May 15;101:27-35. doi: 10.1016/j.ymeth.2015.10.015. Epub 2015 Oct 23.

Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system.

Author information

1
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
2
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan; Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto, Japan.
3
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.

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.

KEYWORDS:

CRISPR; Gene correction; Genome editing; Knock-in; Knock-out; iPS cells

PMID:
26525194
DOI:
10.1016/j.ymeth.2015.10.015
[Indexed for MEDLINE]
Free full text

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center