GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE255nnn/GSE255315/primaryOK200OtherHomo sapiensOtherhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE255315GEOGSEfalseBase editors provoke non-predictable chromosomal translocations and off-target editing - rhAmpBase 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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 sapiens