fig1

Figure 1. Allele-specific Ryr2 gene editing leads to reduction of mutant protein in R176Q/+ mice. (A) Design of AAV vectors lacking insert at the BbsI cloning site (Con, control) and vector with guide RNA (gRNA) insert, packaged with U6 promoter for gRNA and human troponin T (hTNT) promoter for Staphylococcus aureus Cas9 (SaCas9) transcription. (B) Sequences of wild-type and mutant Ryr2 allele, alignment with the gRNA, and most common variants detected in the mutant Ryr2 allele of R176Q/+ mice using amplicon deep sequencing of cDNA at the pathogenic variant site (red). Insertions are shown in light blue. The silent restriction site in the mutant mouse allele (used for genotyping of the mice) is shown in green. (C) Percent of sequence reads with a mutation in the R176Q allele. (D) Percent of sequence reads with a mutation in the WT allele in R176Q/+ mice. (E) Quantification of Ryr2 mRNA levels in WT, control, and gRNA-treated R176Q/+ mice. (F) Western blots and (G) quantification of RyR2 protein levels in WT, control, and gRNA-treated R176Q/+ mice. P-values based on the Mann-Whitney test (C and D), and the Kruskal-Wallis test followed by Dunn’s multiple comparison post-hoc test (E and G).