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

Figures

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  Figure 1.. Inferring the haplotype phase of the de novo mutations responsible for MFM6 in the study subjects. A) To determine whether the de novo mutation (rs121918312-T in exon 3) occurred on the paternal or maternal chromosomes, we used a neighbor SNP, which differed between the father and the mother. Unaffected parents carry different alleles at an intronic SNP rs196330, allowing the inference of the haplotype that carries the disease-causing mutation in the probands. B) Genotypes of the disease-causing mutation (rs121918312) and a neighbor SNP (rs196330) are displayed for study participants, including two probands (subjects #3 and 6; filled black). Red lines and letters represent de novo mutation and disease-causing allele, respectively.

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  Figure 2.. Individualized allele-specific TP-CRISPR strategies for study participants using PAS. A) An allele-specific TP-CRISPR strategy designed for subject #3 is summarized. This strategy utilized one non-allele-specific gRNA targeting an NGG PAM site at Chr10:121410519 and one allele-specific gRNA targeting an NGG PAM site generated by rs3847489. This strategy was expected to excise 6.8KB DNA (involving the TSS) from the mutant BAG3 allele. B) An allele-specific TP-CRISPR strategy for subject #6 utilizes two allele-specific NGG PAM sites generated by rs196290 and rs11199061. The TP-CRISPR strategy for subject #6 was expected to excise 6.7KB DNA from the mutant allele involving the TSS. Black and blue arrows represent non-allele-specific and allele-specific editing, respectively. DNA alleles in green and red indicate mutant and normal alleles at rs121918312.

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  Figure 3.. Allele-specific NMD-CRISPR strategy for MFM6 using the disease-causing mutation as the PSS. Since a nearby NGG PAM site flanks the disease-causing mutation (rs121918312), we designed a CRISPR-Cas9 gRNA using the rs121918312 as the PPS to achieve allele-specific NMD-CRISPR. In this scenario, a gRNA designed to target the mutant allele (19 nucleotide gRNA is displayed) will interact with the mutant allele (A) without mismatches. However, the same gRNA is expected to interact with normal BAG3 with one mismatch (B), potentially leading to mutant-specific NMD.

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  Figure 4.. Molecular consequences of allele-specific NMD-CRISPR. A) MiSeq analysis was performed on mRNA samples from targeted clonal lines from subjects #3 and #6 to evaluate the levels of BAG3 mRNA containing normal and mutant alleles at rs121918312. B) Immunoblot analysis results representing normalized total BAG3 protein are displayed. Three and four independent clonal lines were analyzed for each proband. *, p-value < 0.05 and **, p-value < 0.01. EV, empty vector.

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  Figure 5.. Differentiation into cardiomyocyte from MFM6 patient-derived iPSCs. Immunocytochemistry of CTNT was performed for non-disease control, untargeted, and targeted clonal lines from subject #3 and subject #6 to evaluate the capacity of differentiation into a cardiomyocyte. DAPI staining shows nuclei. The scale bar in the top left panel equals 250 μm.