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

Abstract

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.

Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing

“…Illumina libraries of the parental HepaRG culture were prepared as suggested by the Nextera DNA Flex Library Prep kit (Cat# 20060060) and paired-end sequenced (76 × 2) with 150-cycle kits on a NextSeq 500 instrument until whole genome coverages ~ 30-fold were attained from the processed Illumina dataset (aligned to hg38 by BWA, sorted by Samtools, and marked for duplicates by Picard) (kits and sequencer were from Illumina, San Diego, CA). De-indexing was performed as previously described (Miranda et al 2023 ).…”