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

Authors:
Kurt IC, Zhou R, Iyer S, Garcia SP, Miller BR, Langner LM, Grünewald J, Joung JK
In:
Source: Nat Biotechnol
Publication Date: (2021)
Issue: Jan;39(1): 41-46
Research Area:
Gene Expression
Basic Research
Molecular Biology
Cells used in publication:
HeLa
Species: human
Tissue Origin: cervix
K-562
Species: human
Tissue Origin: blood
U-2 OS
Species: human
Tissue Origin: bone
Platform:
4D-Nucleofector® X-Unit
Experiment

Transfection and electroporation experiments

K562 cells were electroporated using the SF Cell Line Nucleofector X Kit (Lonza), according to the manufacturer’s protocol with 2 × 10^5 cells per nucleofection and 800 ng control or base/prime editor plasmid, 200 ng gRNA or pegRNA plasmid and 83 ng nicking gRNA plasmid (for PE3). U2OS cells were electroporated using the SE Cell Line Nucleofector X Kit (Lonza) with 2 × 10^5 cells and 800 ng control or base/prime editor plasmid, 200 ng gRNA or pegRNA and 83 ng nicking gRNA (for PE3). HeLa cells were electroporated using the SE Cell Line 4D-Nucleofector X Kit (Lonza) with 5 × 10^5 cells and 800 ng control or base/prime editor, 200 ng gRNA or pegRNA and 83 ng nicking gRNA (for PE3).

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.