Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA.
Center for Cancer Research and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA.
Nat Biotechnol. 2021 Jan;39(1):41-46. doi: 10.1038/s41587-020-0609-x. Epub 2020 Jul 20.
CRISPR-guided DNA cytosine and adenine base editors are widely used for many applications 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 activities. 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.
CRISPR 引导的 DNA 胞嘧啶和腺嘌呤碱基编辑器被广泛用于许多应用，但主要是创造 DNA 碱基转换（即嘧啶到嘧啶或嘌呤到嘌呤）。在这里，我们描述了两种碱基编辑器结构的工程设计，它们可以有效地诱导靶向 C 到 G 碱基颠换，同时减少不必要的 C 到 W（W=A 或 T）和插入缺失突变的水平。这两种碱基编辑器（CGBE1）之一，由 RNA 引导的 Cas9 切口酶、源自大肠杆菌的尿嘧啶 DNA N-糖基化酶（eUNG）和先前显示降低 RNA 和 DNA 脱靶编辑活性的大鼠 APOBEC1 胞嘧啶脱氨酶变体（R33A）组成。我们表明，CGBE1 可以有效地诱导 C 到 G 的编辑，特别是在富含 AT 的人类细胞序列环境中。我们还去除了 eUNG 结构域，得到了 miniCGBE1，它降低了插入缺失的频率，但仅适度降低了编辑效率。CGBE1 和 miniCGBE1 可以实现 C 到 G 的编辑，并将为研究和治疗应用优化 C 到 G 碱基编辑器提供基础。
