Small molecule inhibition of ATM kinase increases CRISPR-Cas9 1-bp insertion frequency
Heysol C Bermudez-Cabrera1, Sannie Culbertson1, Sammy Barkal1
1Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.
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.
Insights
Researchers found that inhibiting ATM kinase with KU-60019 significantly boosts 1-bp insertions after CRISPR-Cas9 editing in mammalian cells. This advance enhances control over precise genome editing outcomes, particularly for therapeutic applications.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR-Cas9 gene editing offers predictable outcomes but lacks control over specific repair types.
- Achieving precise 1-bp insertions, crucial for therapeutics, remains challenging.
Purpose of the Study:
- To identify compounds that enhance the frequency of 1-bp insertions following CRISPR-Cas9 editing.
- To improve control over template-free genome editing outcomes.
Main Methods:
- Conducted a small molecule screen to identify modulators of CRISPR-Cas9 repair outcomes.
- Utilized ATM kinase inhibition (small molecule and genetic) across multiple cell lines and Cas9 variants.
- Assessed the impact on 1-bp insertion frequency and overall editing efficiency.
Main Results:
- Identified KU-60019, an ATM kinase inhibitor, that reproducibly increases the fraction of 1-bp insertions.
- ATM inhibition enhanced 1-bp insertion frequency across diverse cell types and target sites.
- Achieved over 80% relative frequency of 1-bp insertions at native human loci, improving correction of pathogenic variants.
Conclusions:
- ATM kinase inhibition provides a novel strategy to enhance 1-bp insertion outcomes in CRISPR-Cas9 editing.
- This method increases precision in template-free genome editing, with potential therapeutic benefits.
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