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

Abstract

CRISPR-guided DNA cytosine and adenine base editors (CBEs and ABEs) are widely used for many applications1–4 but primarily create DNA base transitions (i.e., 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 E. 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.