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Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing

Jianhang Yin1, Rusen Lu1, Changchang Xin1

  • 1The MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Center for Life Sciences, Genome Editing Research Center, Peking University, 100871, Beijing, China.

Nature Communications
|March 9, 2022
PubMed

Insights

CRISPR-Cas9 gene editing can cause unwanted translocations due to repeated DNA cleavage. A new Cas9TX tool prevents this, significantly reducing chromosomal abnormalities and improving editing efficiency for safer gene therapies.

Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Genomics

Background:

  • CRISPR-Cas9 technology is powerful but can induce unintended structural variations.
  • The mechanisms causing these variations, particularly translocations, are not fully understood.
  • Existing strategies to mitigate these byproducts are lacking.

Purpose of the Study:

  • To elucidate the mechanism of CRISPR-Cas9-induced translocations.
  • To develop a novel CRISPR-Cas9 system to minimize chromosomal abnormalities.
  • To enhance the safety and efficiency of gene editing applications.

Main Methods:

  • Investigated the link between repeated Cas9 cleavage and translocation generation.
  • Engineered a Cas9 exo-endonuclease (Cas9TX) by fusing Cas9 with TREX2.
  • Evaluated Cas9TX performance in single-site editing and multiplex gene editing in CAR T cells.

Main Results:

  • High levels of translocations are dependent on repeated cleavage at target sites.
  • Cas9TX significantly suppressed translocation levels compared to standard CRISPR-Cas9.
  • Cas9TX enhanced single-site editing efficiency and reduced large deletions.
  • Multiplex gene editing with Cas9TX in CAR T cells nearly eliminated chromosomal translocations.

Conclusions:

  • Repeated cleavage by Cas9 is a key driver of translocations.
  • Cas9TX offers a strategy to prevent perfect DNA repair, thereby reducing translocations.
  • This approach improves the safety profile of CRISPR-Cas9 for therapeutic applications, especially in complex editing scenarios.

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