Allele-specific CRISPR-Cas9 editing inactivates a single nucleotide variant associated with collagen VI muscular dystrophy

• Published in: Molecular therapy. Nucleic acids 2024-09, Vol.35 (3), p.102269, Article 102269
• Main Authors: Bolduc, Véronique, Sizov, Katherine, Brull, Astrid, Esposito, Eric, Chen, Grace S., Uapinyoying, Prech, Sarathy, Apurva, Johnson, Kory R., Bönnemann, Carsten G.
• Format: Article
• Language: English
• Subjects: allele-specific gene editing; collagen VI; CRISPR-Cas9; guide RNA design; MT: RNA/DNA Editing; muscular dystrophy; Original
• ISSN: 2162-2531; 2162-2531
• DOI: 10.1016/j.omtn.2024.102269

The application of allele-specific gene editing tools can expand the therapeutic options for dominant genetic conditions, either via gene correction or via allelic gene inactivation in situations where haploinsufficiency is tolerated. Here, we used allele-targeted CRISPR-Cas9 guide RNAs (gRNAs) to introduce inactivating frameshifting indels at an SNV in the COL6A1 gene (c.868G>A; G290R), a variant that acts as dominant negative and that is associated with a severe form of congenital muscular dystrophy. We expressed SpCas9 along with allele-targeted gRNAs, without providing a repair template, in primary fibroblasts derived from four patients and one control subject. Amplicon deep sequencing for two gRNAs tested showed that single-nucleotide deletions accounted for the majority of indels introduced. While activity of the two gRNAs was greater at the G290R allele, both gRNAs were also active at the wild-type allele. To enhance allele selectivity, we introduced deliberate additional mismatches to one gRNA. One of these optimized gRNAs showed minimal activity at the WT allele, while generating productive edits and improving collagen VI matrix in cultured patient fibroblasts. This study strengthens the potential of gene editing to treat dominant-negative disorders, but also underscores the challenges in achieving allele selectivity with gRNAs. [Display omitted] Bönnemann and colleagues used CRISPR-Cas9 to install indels to a dominant-negative pathogenic variant in COL6A1, associated with a muscular dystrophy, thereby inactivating the variant gene copy. To avoid inactivating the wild-type copy, they incorporated deliberate modifications to the guide sequence. This approach restored production of collagen VI.