A stress-free strategy to correct point mutations in patient iPS cells

• DOI: 10.1016/j.scr.2021.102332
• Publication Date: 2021-04-08T06:06:07Z
• Authors: Jingli Cai; Elizabeth Kropf; Ya-Ming Hou; Lorraine Iacovitti
• Keywords: 0303 health sciences; Point mutation; 572; QH301-705.5; Induced Pluripotent Stem Cells; Neurosciences; Gene targeting; Medical Biochemistry; Isogenic iPS cells; Article; Clone Cells; 3. Good health; 03 medical and health sciences; Mutation; Humans; Point Mutation; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR-Cas9; Biology (General); CRISPR-Cas Systems; Homologous Recombination

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.