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

• Published in: Stem cell research Vol. 53; p. 102332
• Main Authors: Cai, Jingli, Kropf, Elizabeth, Hou, Ya-Ming, Iacovitti, Lorraine
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 01.05.2021; Elsevier
• Subjects: Clone Cells; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR-Cas Systems - genetics; CRISPR-Cas9; Gene targeting; Homologous Recombination; Humans; Induced Pluripotent Stem Cells; Isogenic iPS cells; Mutation; Point Mutation; Isogenic iPS cells; Gene targeting; CRISPR-Cas9; Point mutation
• DOI: 10.1016/j.scr.2021.102332
• ISSN: 1873-5061; 1876-7753

•A CRISPR-Cas9 gene knock-in method for correction of point mutations or frameshifting.•sgRNA sequences selected from the nearest intron of the mutation providing more flexibility.•Donor vectors containing correct genomic DNA information on the homologous arms and antibiotic selection cassette.•No off-targeting events detected. 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.