APOBEC3 induces mutations during repair of CRISPR-Cas9-generated DNA breaks

Nat Struct Mol Biol. 2018 Jan;25(1):45-52. doi: 10.1038/s41594-017-0004-6. Epub 2017 Dec 11.

Abstract

The APOBEC-AID family of cytidine deaminase prefers single-stranded nucleic acids for cytidine-to-uridine deamination. Single-stranded nucleic acids are commonly involved in the DNA repair system for breaks generated by CRISPR-Cas9. Here, we show in human cells that APOBEC3 can trigger cytidine deamination of single-stranded oligodeoxynucleotides, which ultimately results in base substitution mutations in genomic DNA through homology-directed repair (HDR) of Cas9-generated double-strand breaks. In addition, the APOBEC3-catalyzed deamination in genomic single-stranded DNA formed during the repair of Cas9 nickase-generated single-strand breaks in human cells can be further processed to yield mutations mainly involving insertions or deletions (indels). Both APOBEC3-mediated deamination and DNA-repair proteins play important roles in the generation of these indels. Therefore, optimizing conditions for the repair of CRISPR-Cas9-generated DNA breaks, such as using double-stranded donors in HDR or temporarily suppressing endogenous APOBEC3s, can repress these unwanted mutations in genomic DNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • APOBEC Deaminases
  • CRISPR-Cas Systems*
  • Cytidine / chemistry
  • Cytidine Deaminase / genetics
  • Cytosine Deaminase / chemistry*
  • DNA Breaks, Double-Stranded*
  • DNA Repair*
  • DNA, Single-Stranded
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • INDEL Mutation
  • Mutation*
  • Oligonucleotides / genetics
  • RNA, Small Interfering / metabolism
  • Recombinational DNA Repair
  • Sequence Analysis, DNA

Substances

  • DNA, Single-Stranded
  • Oligonucleotides
  • RNA, Small Interfering
  • Cytidine
  • Cytosine Deaminase
  • APOBEC Deaminases
  • APOBEC3 proteins, human
  • Cytidine Deaminase