Document Type
Article
Publication Date
4-8-2021
Abstract
When studying patient specific induced pluripotent stem cells (iPS cells) as a disease model, the ideal control is an isogenic line that has corrected the point mutation, instead of iPS cells from siblings or other healthy subjects. However, repairing a point mutation in iPS cells even with the newly developed CRISPR-Cas9 technique remains difficult and time-consuming. Here we report a strategy that makes the Cas9 “knock-in” methodology both hassle-free and error-free. Instead of selecting a Cas9 recognition site close to the point mutation, we chose a site located in the nearest intron. We constructed a donor template with the fragment containing the corrected point mutation as one of the homologous recombination arms flanking a PGK-PuroR cassette. After selection with puromycin, positive clones were identified and further transfected with a CRE vector to remove the PGK-PuroR cassette. Using this methodology, we successfully repaired the point mutation G2019S of the LRRK2 gene in a Parkinson Disease (PD) patient iPS line and the point mutation R329H of the AARS1 gene in a Charcot-Marie-Tooth disease (CMT) patient iPS line. These isogenic iPS lines are ideal as a control in future studies.
Recommended Citation
Cai, Jingli; Kropf, Elizabeth; Hou, Ya-Ming; and Iacovitti, Lorraine, "A stress-free strategy to correct point mutations in patient iPS cells" (2021). Department of Biochemistry and Molecular Biology Faculty Papers. Paper 184.
https://jdc.jefferson.edu/bmpfp/184
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.
PubMed ID
33857832
Language
English
Comments
This article is the author’s final published version in Stem Cell Research, Volume 53, May 2021, Article number 102332.
The published version is available at https://doi.org/10.1016/j.scr.2021.102332. Copyright © Cai et al.