CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells

Nat Biotechnol. 2021 Jan;39(1):41-46. doi: 10.1038/s41587-020-0609-x. Epub 2020 Jul 20.

Abstract

CRISPR-guided DNA cytosine and adenine base editors are widely used for many applications1-4 but primarily create DNA base transitions (that is, pyrimidine-to-pyrimidine or purine-to-purine). Here we describe the engineering of two base editor architectures that can efficiently induce targeted C-to-G base transversions, with reduced levels of unwanted C-to-W (W = A or T) and indel mutations. One of these C-to-G base editors (CGBE1), consists of an RNA-guided Cas9 nickase, an Escherichia coli-derived uracil DNA N-glycosylase (eUNG) and a rat APOBEC1 cytidine deaminase variant (R33A) previously shown to have reduced off-target RNA and DNA editing activities5,6. We show that CGBE1 can efficiently induce C-to-G edits, particularly in AT-rich sequence contexts in human cells. We also removed the eUNG domain to yield miniCGBE1, which reduced indel frequencies but only modestly decreased editing efficiency. CGBE1 and miniCGBE1 enable C-to-G edits and will serve as a basis for optimizing C-to-G base editors for research and therapeutic applications.

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-Cas Systems / genetics*
  • Cytidine Deaminase / metabolism
  • Cytosine / metabolism*
  • DNA / genetics
  • DNA / metabolism
  • Gene Editing / methods*
  • Guanine / metabolism
  • HEK293 Cells
  • Humans

Substances

  • Guanine
  • Cytosine
  • DNA
  • Cytidine Deaminase