1. Academic Validation
  2. Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing

Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing

  • Genome Med. 2015 Aug 27;7(1):93. doi: 10.1186/s13073-015-0215-6.
Francis Robert 1 Mathilde Barbeau 2 Sylvain Éthier 3 Josée Dostie 4 Jerry Pelletier 5 6 7
Affiliations

Affiliations

  • 1 Department of Biochemistry, McGill University, Montréal, Québec, H3G 1Y6, Canada. [email protected].
  • 2 Department of Biochemistry, McGill University, Montréal, Québec, H3G 1Y6, Canada. [email protected].
  • 3 Department of Biochemistry, McGill University, Montréal, Québec, H3G 1Y6, Canada. [email protected].
  • 4 Department of Biochemistry, McGill University, Montréal, Québec, H3G 1Y6, Canada. [email protected].
  • 5 Department of Biochemistry, McGill University, Montréal, Québec, H3G 1Y6, Canada. [email protected].
  • 6 Department of Oncology, McGill University, Montréal, Québec, H3G 1Y6, Canada. [email protected].
  • 7 The Rosalind and Morris Goodman Cancer Research Center, McGill University, Montréal, Québec, H3G 1Y6, Canada. [email protected].
Abstract

Background: The ability to modify the genome of any cell at a precise location has drastically improved with the recent discovery and implementation of CRISPR/Cas9 editing technology. However, the capacity to introduce specific directed changes at given loci is hampered by the fact that the major cellular repair pathway that occurs following Cas9-mediated DNA cleavage is the erroneous non-homologous end joining (NHEJ) pathway. Homology-directed recombination (HDR) is far less efficient than NHEJ and makes screening of clones containing directed changes time-consuming and labor-intensive.

Methods: We investigated the possibility of pharmacologically inhibiting DNA-PKcs, a key player in NHEJ, using small molecule inhibitors (NU7441 and KU-0060648), to ameliorate the rates of HDR repair events. These compounds were tested in a sensitive reporter assay capable of simultaneously informing on NHEJ and HDR, as well as on an endogenous gene targeted by Cas9.

Results: We find that NU7441 and KU-0060648 reduce the frequency of NHEJ while increasing the rate of HDR following Cas9-mediated DNA cleavage.

Conclusions: Our results identify two small molecules compatible for use with Cas9-editing technology to improve the frequency of HDR.

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