Oligonucleotide synthesis errors are a source of untoward variation in HDR-mediated gene editing

Single-stranded oligonucleotides (ssODNs) are used as donor templates for therapeutic gene editing by CRISPR-Cas9 cleavage and homology-directed repair (HDR). Although ssODN sequence fidelity is critical to the safety and efficacy of editing, standard quality control methods cannot resolve individual nucleotide errors. By deep sequencing ssODNs from three manufacturers, and amplicons from edited hematopoietic stem/progenitor cells, we find that synthesis errors are present in all ssODNs tested at rates that vary more than two-fold among manufacturers, at positions that are dependent on sequence context. These synthesis errors are propagated into the genome by HDR at frequencies proportional