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

CRISPR J. 2020 Jun;3(3):177-187. doi: 10.1089/crispr.2019.0074.


Multiplexed genome editing with DNA endonucleases has broad application, including for cellular therapies, but chromosomal translocations, natural byproducts of inducing simultaneous genomic breaks, have not been explored in detail. Here we apply various CRISPR-Cas nucleases to edit the T cell receptor alpha and beta 2 microglobulin genes in human primary T cells and comprehensively evaluate the frequency and stability of the resulting translocations. A thorough translocation frequency analysis using three orthogonal methods (droplet digital PCR, unidirectional sequencing, and metaphase fluorescence in situ hybridization) yielded comparable results and an overall translocation rate of ∼7% between two simultaneous CRISPR-Cas9 induced edits. In addition, we show that chromosomal translocations can be reduced when using different nuclease combinations, or by the presence of a homologous single stranded oligo donor for multiplexed genome editing. Importantly, the two different approaches for translocation reduction are compatible with cell therapy applications.

MeSH terms

  • CD4-Positive T-Lymphocytes
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • DNA / genetics
  • Endonucleases / genetics
  • Gene Editing / methods*
  • Genome, Human
  • Humans
  • In Situ Hybridization, Fluorescence
  • Multifactorial Inheritance
  • RNA, Guide
  • Streptococcus pyogenes
  • T-Lymphocytes*
  • Translocation, Genetic*


  • RNA, Guide
  • DNA
  • CRISPR-Associated Protein 9
  • Endonucleases