Personalized allele-specific CRISPR-Cas9 strategies for myofibrillar myopathy 6

Abstract

AbstractMyofibrillar myopathy 6 (MFM6) is a rare childhood-onset myopathy characterized by myofibrillar disintegration, muscle weakness, and cardiomyopathy. The genetic cause of MFM6 is p.Pro209Leu mutation (rs121918312-T) in theBAG3gene, which generates the disease outcomes in a dominant fashion. Since the consequences of theBAG3mutation are strong and rapidly progressing, most MFM6 patients are due tode novomutation. There are no effective treatments for MFM6 despite its well-known genetic cause. Given p.Pro209Leu mutation is dominant, regenerative medicine approaches employing orthologous stem cells in which mutantBAG3is inactivated offer a promising avenue. Here, we developed personalized allele-specific CRISPR-Cas9 strategies capitalizing on PAM-altering SNP and PAM-proximal SNP. In order to identify the disease chromosome carrying thede novomutation in our two affected individuals, haplotype phasing through cloning-sequencing was performed. Based on the sequence differences between mutant and normalBAG3, we developed personalized allele-specific CRISPR-Cas9 strategies to selectively inactivate the mutant allele 1) by preventing the transcription of the mutantBAG3and 2) by inducing nonsense-mediated decay (NMD) of mutantBAG3mRNA. Subsequent experimental validation in patient-derived induced pluripotent stem cell (iPSC) lines showed complete allele specificities of our CRISPR-Cas9 strategies and molecular consequences attributable to inactivated mutantBAG3. In addition, mutant allele-specific CRISPR-Cas9 targeting did not alter the characteristics of iPSC or the capacity to differentiate into cardiomyocytes. Together, our data demonstrate the feasibility and potential of personalized allele-specific CRISPR-Cas9 approaches to selectively inactivate the mutantBAG3to generate cell resources for regenerative medicine approaches for MFM6.