A small molecule ATM kinase inhibitor increases CRISPR/Cas9 1-bp insertion frequency

Abstract: Mutational outcomes following CRISPR/Cas9-nuclease cutting in mammalian cells have recently been shown to be predictable and in certain cases skewed toward single genotypes. However, the ability to control these outcomes remains limited, especially for 1-bp insertions, a common and therapeutically relevant class of repair outcomes. Here, through a small molecule screen, we identify the ATM kinase inhibitor KU-60019 as a compound capable of reproducibly increasing the fraction of 1-bp insertions relative to other Cas9 repair outcomes. KU-60019 increases 1-bp insertion outcome fraction across three human and mouse cell lines, two Cas9 species, and dozens of target sites. Notably, KU-60019 increases the precision of 1-bp insertions past 80% at several native human genomic loci and improves efficiency of correction of pathogenic 1-bp deletion variants. The ability to increase 1-bp insertion frequency adds another dimension to precise template-free Cas9-nuclease genome editing.