Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors

Nat Biotechnol. 2020 May;38(5):620-628. doi: 10.1038/s41587-020-0414-6. Epub 2020 Feb 10.


Cytosine base editors (CBEs) enable targeted C•G-to-T•A conversions in genomic DNA. Recent studies report that BE3, the original CBE, induces a low frequency of genome-wide Cas9-independent off-target C•G-to-T•A mutation in mouse embryos and in rice. Here we develop multiple rapid, cost-effective methods to screen the propensity of different CBEs to induce Cas9-independent deamination in Escherichia coli and in human cells. We use these assays to identify CBEs with reduced Cas9-independent deamination and validate via whole-genome sequencing that YE1, a narrowed-window CBE variant, displays background levels of Cas9-independent off-target editing. We engineered YE1 variants that retain the substrate-targeting scope of high-activity CBEs while maintaining minimal Cas9-independent off-target editing. The suite of CBEs characterized and engineered in this study collectively offer ~10-100-fold lower average Cas9-independent off-target DNA editing while maintaining robust on-target editing at most positions targetable by canonical CBEs, and thus are especially promising for applications in which off-target editing must be minimized.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • CRISPR-Associated Protein 9 / metabolism*
  • Cytosine / metabolism*
  • Escherichia coli / genetics*
  • Gene Editing / economics
  • Gene Editing / methods*
  • HEK293 Cells
  • Humans
  • Mutation
  • Whole Genome Sequencing


  • Cytosine
  • CRISPR-Associated Protein 9