Efficient and Precise CRISPR/Cas9-Mediated MECP2 Modifications in Human-Induced Pluripotent Stem Cells

• Published in: Frontiers in genetics Vol. 10; p. 625
• Main Authors: Le, Thi Thanh Huong, Tran, Ngoc Tung, Dao, Thi Mai Lan, Nguyen, Dinh Dung, Do, Huy Duong, Ha, Thi Lien, Kühn, Ralf, Nguyen, Thanh Liem, Rajewsky, Klaus, Chu, Van Trung
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Media S.A 02.07.2019
• Subjects: CRISPR/Cas9; Genetics; homologous recombination; iPSCs; MECP2 mutations; RETT syndrome; MECP2 mutations; iPSCs; CRISPR/Cas9; homologous recombination; RETT syndrome
• ISSN: 1664-8021; 1664-8021
• DOI: 10.3389/fgene.2019.00625

Patients with Rett syndrome (RTT) have severe mental and physical disabilities. The majority of RTT patients carry a heterozygous mutation in methyl-CpG binding protein 2 (MECP2), an X-linked gene encoding an epigenetic factor crucial for normal nerve cell function. No curative therapy for RTT syndrome exists, and cellular mechanisms are incompletely understood. Here, we developed a CRISPR/Cas9-mediated system that targets and corrects the disease relevant regions of the MECP2 exon 4 coding sequence. We achieved homologous recombination (HR) efficiencies of 20% to 30% in human cell lines and iPSCs. Furthermore, we successfully introduced a MECP2 mutation into the MECP2 gene in human induced pluripotent stem cells (iPSCs). Consequently, using CRISPR/Cas9, we were able to repair such mutations with high efficiency in human mutant iPSCs. In summary, we provide a new strategy for MECP2 gene targeting that can be potentially translated into gene therapy or for iPSCs-based disease modeling of RTT syndrome.