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

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. [Display omitted] •CAST-Seq detects and quantifies designer nuclease-induced chromosomal aberrations•Rearrangements at on-target site occurred with all tested programmable nucleases•Off-target activity is not the only trigger of chromosomal translocations•CAST-Seq enables risk assessment before clinical application of gene-edited product Gene editing is associated with off-target effects that must be evaluated carefully before clinical application. Turchiano et al. established CAST-Seq, a method to identify and quantify CRISPR-Cas- and TALEN-induced chromosomal aberrations. In addition to off-target mediated translocations, they found on-target activity-based chromosomal rearrangements at high frequencies.