Turchiano, G;
Andrieux, G;
Klermund, J;
Blattner, G;
Pennucci, V;
El Gaz, M;
Monaco, G;
... Cathomen, T; + view all
(2021)
Quantitative evaluation of chromosomal rearrangements in gene-edited human stem cells by CAST-Seq.
Cell Stem Cell
, 28
(6)
1136-1147.e5.
10.1016/j.stem.2021.02.002.
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Abstract
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.
| Type: | Article |
|---|---|
| Title: | Quantitative evaluation of chromosomal rearrangements in gene-edited human stem cells by CAST-Seq |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1016/j.stem.2021.02.002 |
| Publisher version: | https://doi.org/10.1016/j.stem.2021.02.002 |
| Language: | English |
| Additional information: | This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions. |
| Keywords: | Science & Technology, Life Sciences & Biomedicine, Cell & Tissue Engineering, Cell Biology, GENOME, CLEAVAGE, TRANSLOCATIONS, RECOMBINATION, REQUIREMENTS, CRISPR/CAS, NUCLEASES, MULTIPLEX, BREAKS |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health > Infection, Immunity and Inflammation Dept |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10137326 |
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