Unbiased whole genome detection of ultrarare off‐target mutations in genome‐edited cell populations by HiFi sequencing

DNA base editors (BEs) composed of a nuclease‐deficient Cas9 fused to a DNA‐modifying enzyme can achieve on‐target mutagenesis without creating double‐strand DNA breaks (DSBs). As a result, BEs generate far less DNA damage than traditional nuclease‐proficient Cas9 systems, which do rely on the creation of DSBs to achieve on‐target mutagenesis. The inability of BEs to create DSBs makes the detection of their undesired off‐target effects very difficult. PacBio HiFi sequencing can efficiently detect ultrarare mutations resulting from chemical mutagenesis in whole genomes with a sensitivity ~1 × 10−8 mutations per base pair. In this proof‐of‐principle study, we evaluated whether this technique could also detect the on‐ and off‐target mutations generated by a cytosine‐to‐thymine (C>T) BE targeting the LacZ gene in Escherichia coli (E. coli). HiFi sequencing detected on‐target mutant allele fractions ranging from ~7% to ~63%, depending on the single‐guide RNA (sgRNA) used, while no on‐target mutations were detected in controls lacking the BE. The presence of the BE resulted in a ~3‐fold increase in mutation frequencies compared to controls lacking the BE, irrespective of the sgRNA used. These increases were mostly composed of C:G>T:A substitutions distributed throughout the genome. Our results demonstrate that HiFi sequencing can efficiently identify on‐ and off‐target mutations in cell populations that have undergone genome editing.