Institute for Transfusion Medicine and Gene Therapy

Designer nucleases (CRISPR-Cas, TALEN), base editors, prime editors, designer nickases, and other gene editors allow scientists to target a specific region within the genome of a particular cell to either knock out or correct a gene. Even though these genome editors are, in general, highly specific, off-target effects have been reported. If off-target activity occurs within a gene or a regulatory region of the genome, unexpected side effects can occur, which, in a worst-case scenario, are tumorigenic. Therefore, the activity and specificity of each designer genome modifier has to be validated thoroughly before any clinical application.

Our lab developed several methods to detect off-target effects of genome editors, incl. CAST-Seq, an unbiased and highly sensitive assay that identifies chromosomal rearrangements triggered by the expression of genome editors and that is able to nominate off-targets sites (Turchiano et al. 2021 Cell Stem Cell, Rhiel et al. 2023 Frontiers in Genome Editing; Patents US11319580B2/EP3856928B1). We successfully used CAST-Seq to nominate off-target sites as well as to identify chromosomal aberrations at on-target and off-target sites in a variety of different primary cells that were edited ex vivo or in vivo. To validate CAST-Seq results we use a combination of next generation sequencing (NGS)-based amplicon sequencing (short-read Illumina and long-read PacBio/Nanopore) and/or rhAmpSeq™.