GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE329nnn/GSE329615/primary200OKOtherHomo sapiensOtherhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE329615GEOGSEfalseBase editors provoke non-predictable chromosomal translocations and off-target editing (rhAmp part2)Base editing has emerged as a promising alternative to CRISPR-Cas nuclease-based genome editing, in part because of its promise of reduced genotoxicity. Here, we performed a comprehensive genome-wide analysis of base editing-induced off-target effects. Primary human T cells were edited at three genomic loci (CCR5, EMX1, FANCF) using either CRISPR-Cas9 nuclease, an adenine base editor (ABE8e), one of three cytosine base editors (CBEs), or Cas9 nickase. We used CAST-Seq to identify gross chromosomal aberrations and off-target sites (OTs), followed by rhAmp-Seq to evaluate on-target activity (44-84% modified alleles) and mutagenic effects at >100 putative OTs. On-target CAST-Seq reads showed large (>200 bp) aberrations ranging from 45% for Cas9 to 5% for ABE, 2-7% for CBEs, and 0% for nickase-edited cells. While ABE8e editing induced few translocations, more than 10% of the alleles were modified in 17 of the 40 identified OTs. In contrast, chromosomal translocation events in some CBE-treated cells exceeded that found in Cas9-edited T cells by 3-fold. Furthermore, extensive C-to-T conversion (≤99% of alleles) and mutagenesis (≤7% of alleles) were detected in T cells edited with a hyperactive CBE, whereas recently developed CBE variants (TadCBEd or CBE-T1.52) showed only marginal OT activity. In conclusion, our study revealed the genotoxic potential of ABE8e and the hyperactive evoCDA1-BE4max. These base editors induced mutagenic OT editing at sites that were not altered by CRISPR-Cas9 nucleases complexed with the same gRNA, suggesting that the mechanism of gRNA-dependent OT activity of base editors is different from that of CRISPR-Cas nucleases.2026/09/28GSE329615GSM9707651GSM9707640GSM9707652GSM9707641GSM9707653GSM9707642GSM9707654GSM9707643GSM9707650GSM9707648GSM9707649GSM9707644GSM9707645GSM9707646GSM970764724676329615Homo sapiens