CRISPR-Cas9 provides a tool to treat autosomal dominant disease by non-homologous end joining (NHEJ) gene disruption of the mutant allele. In order to discriminate between wild-type and mutant alleles, Streptococcus pyogenes Cas9 (SpCas9) must be able to detect a single nucleotide change. Allele-specific editing can be achieved by using either a guide-specific approach, in which the missense mutation is found within the guide sequence, or a protospacer-adjacent motif (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 (transforming growth factor β-induced) corneal dystrophies, it is neither possible nor realistic to target each mutation individually. In this study, we demonstrate allele-specific CRISPR gene editing independent of the disease-causing mutation that is capable of achieving complete allele discrimination, and we propose it as a targeting approach for autosomal dominant disease. Our approach utilizes natural variants in the target region that contain a PAM on one allele that 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 personalized manner.
- allele specificity
- autosomal dominant disease
- gene therapy
- personalised medicine
Christie, K., Robertson, L., Conway, C., Blighe, K., Dedionisio, L., Chao-Shern, C., Kowalczyk, A., Marshall, J., Turnbull, D., Nesbit, M. A., & Moore, T. C. B. (2020). Mutation-independent allele-specific editing by CRISPR-Cas9, a novel approach to treat autosomal dominant disease: Mutation-independent allele-specific CRISPR editing. Molecular Therapy, 28(8), 1846-1857. https://doi.org/10.1016/j.ymthe.2020.05.002