Small molecule inhibition of ATM kinase increases CRISPR-Cas9 1-bp insertion frequency

Nat Commun. 2021 Aug 25;12(1):5111. doi: 10.1038/s41467-021-25415-8.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Ataxia Telangiectasia Mutated Proteins / antagonists & inhibitors*
  • Ataxia Telangiectasia Mutated Proteins / genetics
  • Ataxia Telangiectasia Mutated Proteins / metabolism*
  • CRISPR-Cas Systems / drug effects*
  • Cell Line
  • Gene Editing
  • Humans
  • Morpholines / pharmacology*
  • Mutagenesis, Insertional / drug effects*
  • Protein Kinase Inhibitors / pharmacology*
  • Sequence Deletion / drug effects
  • Thioxanthenes / pharmacology*

Substances

  • 2-(2,6-dimethylmorpholin-4-yl)-N-(5-(6-morpholin-4-yl-4-oxo-4H-pyran-2-yl)-9H-thioxanthen-2-yl)acetamide
  • Morpholines
  • Protein Kinase Inhibitors
  • Thioxanthenes
  • ATM protein, human
  • Ataxia Telangiectasia Mutated Proteins

Associated data

  • figshare/10.6084/m9.figshare.12844577
  • figshare/10.6084/m9.figshare.12844625
  • figshare/10.6084/m9.figshare.12844631
  • figshare/10.6084/m9.figshare.14046581