BioE Seminar: Precise Gene Editing of Mutant Alleles Using CRISPR-Cas9 Ribonucleoprotein Complexes

Abstract: Genome editing and stem cell technologies have given biomedical engineers new raw materials and tools to make therapeutics. These technologies offer tremendous potential to rewrite the human genome and cellular composition of our bodies. Using cell/tissue platforms derived from patient-specific pluripotent stem cells (PSCs), I will describe the development novel genome surgery strategies to edit pathological mutations within the body. We have designed strategies to(1) cut and destroy "poison pill" dominant mutations and (2) correct mutant alleles in recessive disorders. First, we observe that mutant allele specific editing is possible without disruption of the normal 'healthy' allele, within a retinal disease PSC model for bestrophinopathy. We also surprisingly find different edited mutant alleles within PSC-derived retinal pigmented epithelial cells versus their isogenic PSCs. Because the differently edited alleles result in robust functional rescue of bestrophinopathic phenotypes, this work charts a new translational path for the development of gene editing approaches for these disorders. Second, using our CRISPR S1mplex and ArrayEdit technologies, we demonstrate that gene correction of both mutant alleles within an autosomal recessive case of Pompe disease rescues the disease phenotype more effectively than correcting either mutant allele. For somatic editing within the body, this work informs new design principles.