Detect-seq reveals out-of-protospacer editing and target-strand editing by cytosine base editors

Nat Methods. 2021 Jun;18(6):643-651. doi: 10.1038/s41592-021-01172-w. Epub 2021 Jun 7.

Abstract

Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, their genome-wide specificity remains poorly understood. Here we report Detect-seq for the evaluation of CBE specificity. It enables sensitive detection of CBE-induced off-target sites at the genome-wide level. Detect-seq leverages chemical labeling and biotin pulldown to trace the editing intermediate deoxyuridine, thereby revealing the editome of CBE. In addition to Cas9-independent and typical Cas9-dependent off-target sites, we discovered edits outside the protospacer sequence (that is, out-of-protospacer) and on the target strand (which pairs with the single-guide RNA). Such unexpected off-target edits are prevalent and can exhibit a high editing ratio, while their occurrences exhibit cell-type dependency and cannot be predicted based on the sgRNA sequence. Moreover, we found out-of-protospacer and target-strand edits nearby the on-target sites tested, challenging the general knowledge that CBEs do not induce proximal off-target mutations. Collectively, our approaches allow unbiased analysis of the CBE editome and provide a widely applicable tool for specificity evaluation of various emerging genome editing tools.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems
  • Cytosine / metabolism*
  • Gene Editing / methods*
  • Humans
  • MCF-7 Cells
  • Mutation
  • RNA / genetics
  • Whole Genome Sequencing

Substances

  • RNA
  • Cytosine