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Updated: Jul 9, 2025

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter
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Cas9 is mostly orthogonal to human systems of DNA break sensing and repair

Ekaterina A Maltseva1, Inna A Vasil'eva1, Nina A Moor1

  • 1SB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia.

Plos One
|November 29, 2023
PubMed

Insights

The CRISPR/Cas9 gene editing tool functions independently of human DNA repair systems. Cas9 effectively shields DNA breaks from repair sensors, indicating its orthogonality to cellular repair mechanisms.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • The CRISPR/Cas9 system is a key gene editing technology.
  • Cas9 activity can be influenced by DNA damage and repair proteins.
  • Understanding this interaction is crucial for optimizing gene editing applications.

Purpose of the Study:

  • To investigate the interplay between Streptococcus pyogenes Cas9 and human DNA repair factors.
  • To determine if DNA repair proteins modulate Cas9 activity.
  • To assess if Cas9 is regulated by natural DNA break sensing pathways.

Main Methods:

  • Studied interactions between Cas9 and DNA repair proteins like PARP1, PARP2, Ku, Ligase I, RPA, and YB-1.
  • Assessed the impact of these proteins on Cas9-induced DNA breaks (DSBs and SSBs).
  • Evaluated Cas9 activity and gene editing efficiency in cellular models.

Main Results:

  • Cas9 activity was not significantly affected by the tested DNA repair factors.
  • Cas9 effectively shielded DNA double-strand breaks (DSBs) and single-strand breaks (SSBs) from cellular sensors.
  • Poly(ADP-ribosyl)ation of Cas9 by PARP2 did not alter its activity.
  • Cas9-mediated gene editing was independent of poly(ADP-ribose) polymerase 1 (PARP1) in cells.

Conclusions:

  • CRISPR/Cas9 operates largely independently of human DNA damage sensing and repair pathways.
  • Cas9 can be considered an orthogonal system to natural cellular regulation of DNA breaks.
  • This orthogonality suggests robustness for Cas9 gene editing applications.

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