Sensitive and unbiased genome-wide profiling of base-editor-induced off-target activity with CHANGE-seq-BE [Hybrid Capture Sequencing for CBE and ABE]

To assess genome-wide off-target activity of base editors and Cas9 nucleases identified by CHANGE-seq-BE, Digenome-seq, and CHANGE-seq, we performed hybrid capture sequencing for five therupatic loci (B2M, CBLB, CD7, CIITA, PDCD1) in human primary T-cells and PCSK9 in human hepatocytes. We observed high on-target editing for ABE, CBE, Cas9 mRNA edited cells and potential off-targets confirmed by hybrid capture sequencing. Our results demonstrate that CHANGE-seq-BE is highly sensitive than Digenome-seq to detect more bona fide off-targets with cellular activity ranging from 0.5% to 92.2%. Moreover, ABE exhibit more off-targets than Cas9 under same delivery conditions. Overall design: Hybrid capture sequencing to validate bona fide off-targets sites nominated by CHANGE-seq-BE, CHANGE-seq, and Digenome-seq in primary human T-cells and hepatocytes.