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. Small molecule or genetic ATM inhibition increases 1-bp insertion outcome fraction across three human and mouse cell lines, two Cas9 species, and dozens of target sites, although concomitantly reducing the fraction of edited alleles. Notably, KU-60019 increases the relative frequency of 1-bp insertions to over 80% of edited alleles at several native human genomic loci and improves the efficiency of correction for pathogenic 1-bp deletion variants. The ability to increase 1-bp insertion frequency adds another dimension to precise template-free Cas9-nuclease genome editing.
Highlights
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
In order to address whether KU-60019 increases 1-bp insertion frequency through Ataxia Telangiectasia Mutated (ATM) inhibition and not an alternative pathway, we investigated the effects of Atm knockout on Cas[9] mutational outcomes. mouse embryonic stem cell (mESC) expressing a GFP transgene were dosed with Cas[9] and a guide RNAs (gRNAs) targeting Atm or a non-targeting control
In this work, we show that the ATM inhibitor KU-60019 reproducibly skews the mutational outcomes of both Streptococcus Pyogenes Cas9 (SpCas9)-NG and KKH-SaCas[9] toward 1-bp insertions across dozens of native and synthetic sites in three human and mouse cell lines
Summary
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. 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 Cas[9] repair outcomes. Small molecule or genetic ATM inhibition increases 1-bp insertion outcome fraction across three human and mouse cell lines, two Cas[9] species, and dozens of target sites, concomitantly reducing the fraction of edited alleles. Ku complexes with DNA-PKcs, A DNA-dependent protein kinase whose autophosphorylation recruits and directs DNA ligase IV, XLF, and other accessory factors to the DSB6. This complex, called the paired-end complex, ligates the broken DNA ends back together. NHEJ tends to be the fastest method of repairing Cas9-induced DSBs and can complete a DSB repair as soon as 30 min after the cut[7]
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