Mutation-Independent Allele-Specific Editing by CRISPR-Cas9, a Novel Approach to Treat Autosomal Dominant Disease.

Abstract CRISPR-Cas9 provides a tool to treat autosomal dominant disease by NHEJ gene disruption of the mutant allele. In order to discriminate between wild-type and mutant alleles, SpCas9 must be able to detect a single nucleotide change. Allele-specific editing can be achieved by employing either a guide-specific approach, in which the missense mutation is found within the guide sequence; or a PAM-specific approach, in which the missense mutation generates a novel PAM. While both approaches have been shown to offer allele-specificity in certain contexts, in cases where numerous missense mutations are associated with a particular disease, such as TGFBI corneal dystrophies, it is neither possible nor realistic to target each mutation individually. Here we demonstrate allele-specific CRISPR gene editing independent of the disease-causing mutation which is capable of achieving complete allele discrimination and we propose it as a targeting approach for autosomal dominant disease. Our approach utilises natural variants in the target region that contain a PAM on one allele which lies in cis with the causative mutation, removing the constraints of a mutation-dependent approach. Our innovative patient-specific guide design approach takes into account the patient’s individual genetic make-up allowing on and off target activity to be assessed in a personalised manner.