Highly efficient stem cell genome editing to study the genetic basis of modern humans and disease-relevant mutations
1Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
CRISPR-Cas9 genome editing has revolutionized molecular biology as it allows targeted modification of a gene of interest. This helps to investigate the molecular basis of life, the genetic basis of modern humans, as well as disease-relevant mutations. The first step in CRISPR-Cas9-mediated genome editing is the cleavage of target DNA sequences that are complementary to spacer sequences in the respective CRISPR gRNA. Cellular repair of CRISPR induced DNA double-strand breaks is dominated by error prone end joining pathways that outcompete inefficient homology-directed repair (HDR) that would allow precise introduction of a mutation present in a supplied synthetic DNA donor. I will present methods to increase CRISPR-Cas9 mediated cleavage of DNA (up to 1000-fold) by an engineered gRNA and to increase HDR-dependent precise genome editing (up to 90%) by modulation of DNA repair pathways.