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Detection and Modulation of DNA Translocations During Multi-Gene Genome Editing in T Cells

Anne Bothmer1,2, Kenneth W Gareau1, Hayat S Abdulkerim1,3

  • 1Editas Medicine, Cambridge, Massachusetts, USA.

The CRISPR Journal
|June 26, 2020
PubMed

Insights

Multiplexed genome editing using CRISPR-Cas9 can cause chromosomal translocations in T cells. Researchers found ways to reduce these translocations, making the technology safer for cell therapies.

Area of Science:

  • Genomics
  • Molecular Biology
  • Cell Therapy

Background:

  • Multiplexed genome editing enables simultaneous genetic modifications.
  • Chromosomal translocations are a potential risk of inducing multiple DNA breaks.
  • The frequency and stability of these translocations are not well understood.

Purpose of the Study:

  • To evaluate the frequency and stability of chromosomal translocations resulting from multiplexed genome editing.
  • To investigate methods for reducing translocation rates in CRISPR-Cas edited cells.
  • To assess the compatibility of translocation reduction strategies with cell therapy.

Main Methods:

  • CRISPR-Cas nucleases were used to edit T cell receptor alpha and beta 2 microglobulin genes in human primary T cells.
  • Translocation frequency was analyzed using droplet digital PCR, unidirectional sequencing, and metaphase fluorescence in situ hybridization.
  • The impact of different nuclease combinations and oligo donors on translocation rates was assessed.

Main Results:

  • Simultaneous CRISPR-Cas9 edits resulted in an approximate 7% translocation rate.
  • Different nuclease combinations and the presence of oligo donors reduced chromosomal translocations.
  • The identified reduction methods are compatible with cell therapy applications.

Conclusions:

  • Multiplexed genome editing can lead to chromosomal translocations, but at a manageable frequency.
  • Strategies exist to mitigate translocation risks during multiplexed genome editing.
  • These findings support the advancement of CRISPR-Cas based cell therapies.

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