Quantitative evaluation of chromosomal rearrangements in gene-edited human stem cells by CAST-Seq

Cell Stem Cell. 2021 Jun 3;28(6):1136-1147.e5. doi: 10.1016/j.stem.2021.02.002. Epub 2021 Feb 23.


Genome editing has shown great promise for clinical translation but also revealed the risk of genotoxicity caused by off-target effects of programmable nucleases. Here we describe chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing (CAST-Seq), a preclinical assay to identify and quantify chromosomal aberrations derived from on-target and off-target activities of CRISPR-Cas nucleases or transcriptional activator-like effector nucleases (TALENs), respectively, in human hematopoietic stem cells (HSCs). Depending on the employed designer nuclease, CAST-Seq detected translocations in 0%-0.5% of gene-edited human CD34+ HSCs, and up to 20% of on-target loci harbored gross rearrangements. Moreover, CAST-Seq detected distinct types of chromosomal aberrations, such as homology-mediated translocations, that are mediated by homologous recombination and not off-target activity. CAST-Seq is a sensitive assay able to identify and quantify unintended chromosomal rearrangements in addition to the more typical mutations at off-target sites. CAST-Seq analyses may be particularly relevant for therapeutic genome editing to enable thorough risk assessment before clinical application of gene-edited products.

Keywords: CRISPR-Cas; chromosomal aberrations; chromosomal rearrangements; clinical risk assessment; designer nucleases; gene editing; off-target activity; off-target effects; programmable nucleases; translocations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems* / genetics
  • Chromosome Aberrations
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Gene Editing*
  • Humans
  • Stem Cells