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Cell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR-Cas9

G Cullot1, J Boutin1,2, S Fayet1

  • 1Bordeaux University, INSERM, BRIC, U1312, F-33000, Bordeaux, France.

Nature Communications
|July 10, 2023
PubMed

Insights

New Fluorescence-Assisted Megabase-scale Rearrangements Detection (FAMReD) systems accurately detect gene editing outcomes. Cell cycle arrest during CRISPR-Cas9 editing reduces loss of heterozygosity (LOH) risk in clinical applications.

Area of Science:

  • Genetics
  • Molecular Biology
  • Genomics

Background:

  • CRISPR-Cas9 gene editing offers precise genomic modification with clinical potential.
  • Standard PCR methods underestimate on-target genotoxicity from gene editing.
  • Sensitive detection of complex gene editing outcomes is crucial.

Purpose of the Study:

  • To develop sensitive detection methods for complex gene editing outcomes.
  • To quantify megabase-scale loss of heterozygosity (LOH) after gene editing.
  • To investigate factors influencing LOH frequency.

Main Methods:

  • Development and application of two complementary Fluorescence-Assisted Megabase-scale Rearrangements Detection (FAMReD) systems.
  • Detection, quantification, and cell sorting of edited cells exhibiting LOH.
  • Analysis of LOH frequency in relation to cell division rate and p53 status.

Main Results:

  • FAMReD systems detect rare, complex chromosomal rearrangements induced by Cas9.
  • Loss of heterozygosity (LOH) frequency is dependent on cell division rate and p53 status.
  • Cell cycle arrest during editing significantly suppresses LOH without affecting editing efficiency.

Conclusions:

  • FAMReD provides a sensitive tool for analyzing gene editing products.
  • Cell cycle arrest is a viable strategy to mitigate LOH risk during gene editing.
  • Clinical trial protocols should consider p53 status and proliferation rates for safer gene editing applications.

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