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Quantitative Evaluation of Chromosomal Rearrangements in Primary Gene-Edited Human Stem Cells by Preclinical CAST-Seq

44 Pages Posted: 9 Apr 2020 Publication Status: Published

See all articles by Giandomenico Turchiano

Giandomenico Turchiano

University of Freiburg - Institute for Transfusion Medicine and Gene Therapy

Geoffroy Andrieux

University of Freiburg

Georges Blattner

University of Freiburg - Institute for Transfusion Medicine and Gene Therapy

Valentina Pennucci

University of Freiburg - Institute for Transfusion Medicine and Gene Therapy

Julia Klermund

University of Freiburg - Institute for Transfusion Medicine and Gene Therapy

Gianni Monaco

University of Freiburg - Institute for Transfusion Medicine and Gene Therapy

Sushmita Poddar

University of Freiburg - Institute for Transfusion Medicine and Gene Therapy

Claudio Mussolino

University of Freiburg - Institute for Transfusion Medicine and Gene Therapy

Tatjana I. Cornu

University of Freiburg - Institute for Transfusion Medicine and Gene Therapy

Melanie Boerries

University of Freiburg

Toni Cathomen

University of Freiburg - Institute for Transfusion Medicine and Gene Therapy

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Abstract

Genome editing with programmable nucleases has shown great promise for clinical translation but also revealed the risk of genotoxicity caused by chromosomal translocations or the insertion of mutations at off-target sites. Here, we describe CAST-Seq, an innovative assay to identify and quantify chromosomal aberrations derived from on- and off-target activities of CRISPR-Cas nucleases or TALENs. CAST-Seq also detected novel types of chromosomal rearrangements, including homology-mediated translocations that are mediated by homologous recombination. Depending on the employed designer nuclease, translocations occurred in 0–0.5% of gene-edited human stem cells and some 20% of target loci harbored gross aberrations. In conclusion, CAST-Seq analyses are particularly relevant for therapeutic editing of stem cells to enable a thorough risk assessment before clinical application of gene editing products.

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

Suggested Citation

Turchiano, Giandomenico and Andrieux, Geoffroy and Blattner, Georges and Pennucci, Valentina and Klermund, Julia and Monaco, Gianni and Poddar, Sushmita and Mussolino, Claudio and Cornu, Tatjana I. and Boerries, Melanie and Cathomen, Toni, Quantitative Evaluation of Chromosomal Rearrangements in Primary Gene-Edited Human Stem Cells by Preclinical CAST-Seq. Available at SSRN: https://ssrn.com/abstract=3565007 or http://dx.doi.org/10.2139/ssrn.3565007
This version of the paper has not been formally peer reviewed.

Giandomenico Turchiano

University of Freiburg - Institute for Transfusion Medicine and Gene Therapy

Germany

Geoffroy Andrieux

University of Freiburg

Fahnenbergplatz
Freiburg, D-79085
Germany

Georges Blattner

University of Freiburg - Institute for Transfusion Medicine and Gene Therapy

Germany

Valentina Pennucci

University of Freiburg - Institute for Transfusion Medicine and Gene Therapy

Germany

Julia Klermund

University of Freiburg - Institute for Transfusion Medicine and Gene Therapy

Germany

Gianni Monaco

University of Freiburg - Institute for Transfusion Medicine and Gene Therapy

Germany

Sushmita Poddar

University of Freiburg - Institute for Transfusion Medicine and Gene Therapy

Germany

Claudio Mussolino

University of Freiburg - Institute for Transfusion Medicine and Gene Therapy

Germany

Tatjana I. Cornu

University of Freiburg - Institute for Transfusion Medicine and Gene Therapy

Germany

Melanie Boerries

University of Freiburg

Fahnenbergplatz
Freiburg, D-79085
Germany

Toni Cathomen (Contact Author)

University of Freiburg - Institute for Transfusion Medicine and Gene Therapy ( email )

Germany

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