Homo sapiens Targeted Locus (Loci)

Prime editing systems have enabled the incorporation of precise edits within a genome without introducing double strand breaks. Previous studies defined an optimal primer binding site (PBS) length for the pegRNA of ~13 nucleotides depending on the sequence composition. However, characterization of optimal PBS length has been based evaluated on prime editing outcomes using plasmid or lentiviral expression systems. In this study we demonstrate that forin the case of prime editor ribonucleoprotein complexes, the auto-inhibitory interaction of the PBS-spacer region in the pegRNA affects target recognition, as well as the pegRNA binding loading efficiency. We show that destabilizing this auto-inhibitory interaction by reducing the complementarity between the PBS-spacer region enhances prime editing efficiency. In the case of end protected pegRNAs, shorter PBS lengths with a PBS-target strand melting temperature near 37C are optimal. Additionally, a transient cold shock treatment of the cells post PE-pegRNA delivery further increases prime editing outcomes of pegRNAs with shorter PBS lengths. Finally, we show that prime editor ribonucleoprotein complexes programmed withend protected, chemically synthesized pegRNAs designed using with these refined parameters are able to efficiently correct disease relevant mutations in patient derived fibroblasts and install precise edits in primary human T cells.