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

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. Overall design: CD34 positive HSPC were treated with CRISPR/CAS9 or TALEN nucleases. In total 30 samples treated with nulceases were analysed together with their respective untreated controls (one control per sample). The same non-treated sample was used whenever we analysed the Wild-Type or the High Fidelity (HiFi) nuclease. Several days post treatment were considered: 1, 4 and 14.