Abstract Defining off-target profiles of gene-editing nucleases and CRISPR base editors remains an important challenge for use of these technologies, therapeutic or otherwise. Existing methods can identify off-target sites induced by these gene editors on an individual genome but are not designed to account for the broad diversity of genomic sequence variation that exists within populations of humans or other organisms. Here we describe OligoNucleotide Enrichment and sequencing ( ONE-seq ), a novel in vitro method that leverages customizable, high-throughput DNA synthesis technology instead of purified genomic DNA ( gDNA ) from individual genomes to profile gene editor off-target sites. We show that ONE-seq matches or exceeds the sensitivity of existing single-genome methods for identifying bona fide CRISPR-Cas9 off-target sites in cultured human cells and in vivo in a liver-humanized mouse model. In addition, ONE-seq outperforms existing best-in-class single-genome methods for defining off-target sites of CRISPR-Cas12a nucleases, cytosine base editors ( CBE s), and adenine base editors ( ABE s), unveiling previously undescribed bona fide off-target sites for all these editors in human cells. Most importantly, we leveraged ONE-seq to generate the first experimentally-derived population-scale off-target profiles for Cas9 nucleases that define the impacts of sequence variants from &gt;2,500 individual human genome sequences in the 1000 Genomes Project database. Notably, some of the variants we identified that lead to increased mutation frequencies at off-target sites are enriched in specific human populations. We validated that novel population-specific, variant-sensitive off-target sites nominated by ONE-seq in vitro can show increased frequencies of mutations in human lymphoblastoid cells ( LCL s) harboring these sequence variants. Collectively, our results demonstrate that ONE-seq is a highly sensitive off-target nomination method that can uniquely be used to identify population subgroup-linked differences in off-target profiles of gene editors. ONE-seq provides an important new pathway by which to assess the impacts of global human genetic sequence diversity on the specificities of gene editors, thereby enabling a broader and more all-inclusive approach for profiling off-target effects of these transformative therapeutic technologies.

Global-scale CRISPR gene editor specificity profiling by ONE-seq identifies population-specific, variant off-target effects