A Robust Gene Editing Approach for Large Genomic Insertions for Monogenetic Diseases

Stanford scientists have developed a gene editing approach that can correct hundreds of hotspot mutations in the COL7A1 gene locus by adapting the Easi-CRISPR strategy with only one set of single guide RNAs (sgRNA). COL7A1 mutations lead to recessive dystrophic epidermolysis bullosa (RDEB) that causes the skin to blister and shear.

Current CRISPR strategies require the sgRNAs to be near the mutation sites, requiring in vitro and in vivo validation for each reagent used for mutations that are not localized to a common site. CRISPR/Cas9 also requires the use of double stranded DNA (dsDNA) which has size limitations, low integration, and potential for unintended loci insertion. To circumvent these issues, Stanford researchers have modified the Easi-CRISPR strategy to treat hotspot mutations in the COL7A1 gene in patient cells. Stanford scientists adapted Easi-CRISPR, traditionally used for knockout mouse models, to correct mutations by inserting large (~2.7 kb) single-stranded DNA (ssDNA) into a target locus on the COL7A1 gene from patient cells. This method maintains high rates of large donor DNA integration, bypassing the need to be close to the mutations. In addition, the nature of the ssDNA greatly increases insertion specificity into the target locus. This reduces the likelihood for potential oncogenic changes that could affect RDEB patients who are already predisposed to developing squamous cell carcinoma.

The ability to insert large kilobases of genes can allow for the treatment of at least 33% of RDEB patients with fewer reagents, saving time and resources. This gene therapy approach provides a valuable strategy for monogenetic diseases with proof of concept established in patient cells with RDEB.

Stage of Development:

Proof of concept – in vitro data from patient cells