NEW YORK (GenomeWeb) – Scientists from the UK have exploited single nucleotide polymorphisms in alleles that cause corneal dystrophy to edit the genes using CRISPR/Cas9.
While CRISPR/Cas9 genome editing can be programmed to target many sections of the genome, the technique will only make a double-stranded break if the guide RNA matches up next to a three-base-pair section called a protospacer adjacent motif (PAM). For Cas9 enzymes from Streptococcus pyogenes commonly used in genome editing applications, PAMs take the form of NGG.
The scientists, led by the University of Ulster's Tara Moore and Andrew Nesbit, recognized that SNPs could create a PAM not found in wild-type alleles. In cases where the SNP corresponds to a deleterious allele, the resulting PAM could provide a point of entry for editing-based gene therapy.
In the paper, published yesterday in Nature Gene Therapy, the researchers singled out the gene KRT12, where a dominant-negative SNP causes Meesmann's epithelial corneal dystrophy, a disease characterized by opaque build-up in the cornea. The L132P mutation changes an "AAG" sequence to "AGG," satisfying the requirements of an S. pyogenes Cas9 PAM.
While CRISPR/Cas9 editing can result in two different repair mechanisms, either one would lead to a successful intervention, the authors said. Genome repair by non-homologous end joining (NHEJ) would result in a frameshift mutation, preventing expression, or it would be repaired by homology directed repair with a template of the wild-type allele. "Both outcomes could be considered a therapeutic success," the authors wrote, adding that damage to one KRT12 allele does not seem to negatively affect corneal integrity.
The authors showed that they could edit the gene at the novel PAM created by the SNP. In a mouse model, they demonstrated successful in vivo editing of the allele, where five of the 13 mice sequenced exhibited deletions ranging between 18 and 53 nucleotides.
While the study focused on the L132P mutation, the authors said that many SNPs can create new PAMs.
"Our analysis of the dominant-negative missense mutations that cause a range of different corneal dystrophies reveals that up to one third result in a novel [S. pyogenes] PAM site that could be potentially targeted by this approach," Nesbit told GenomeWeb in an email.
And the results could apply to diseases other than corneal dystrophies. "The potential exists for developing therapies for a wide range of heterozygous disease-causing SNPs, whether to knockout the mutant allele, as demonstrated here using NHEJ, or to repair the mutant allele, using similar methods already published using homology-directed repair," the authors said.