Abstract
Several gene editing tools have entered the clinic, representing varied options for eliminating or correcting mutations. Although gene editing by homologous recombination (HR) can potentially accomplish any type of gene edit (insertions, deletions, and replacements), as the outcome is defined by a recombinant repair template, gene editing enzymes that support efficient HR are rare. ARCUS nucleases, engineered from the homing endonuclease I-CreI, have programmable sequence specificity and support precise, high-frequency transgene insertion. In this study, we demonstrate that the 3' overhangs that ARCUS nucleases generate when cutting DNA are key to triggering high rates of HR. We show that a single editor can be used to accomplish the full range of currently understood DNA editing approaches, allowing all combinations of single base changes, introducing small, specific deletions, small and large insertions, and the ability to replace large segments of genomic DNA with efficiencies ranging from 60% to 90% in lymphocytes. ARCUS also supports precise, efficient insertion (30%-40%) in noncycling hepatocytes via nonclassical HR pathways. Collectively, this work characterizes a flexible and efficient gene insertion system for potential therapeutic use.