小柯机器人

哈佛医学院J. Keith Joung和Julian Grünewald小组合作在单碱基编辑工具研究中取得进展。他们开发出CRISPR C-G碱基颠换工具，能够编辑诱导目标在人类细胞中DNA颠换。相关论文于2020年7月20日发表于《自然—生物技术》杂志。

研究团队通过对两种碱基编辑器结构的改造，有效地诱导目标C-G碱基颠换，同时减少了不必要的C-W(W = A或T)和插入缺失突变（indel)。其中一个C-G碱基编辑器(CGBE1)，包含RNA介导 Cas9 nickase，以大肠杆菌为基础的尿嘧啶DNA糖基化酶 (eUNG)和以大鼠APOBEC为基础的胞嘧啶核苷脱氨酶突变体（R33A）（过去工作证明该突变体能够降低RNA和DNA编辑中的脱靶效应）。

研究团队表明，CGBE1可以有效地诱导C到G的编辑，尤其是在人类细胞中富含AT的序列中。通过删除CGBE1中的eUNG域，课题组得到了miniCGBE1，降低了插入缺失突变的频率，而编辑效率仅略有减少。CGBE1和miniCGBE1成功实现C-G编辑，并为优化C-G碱基编辑的研究和治疗应用提供了基础。

研究人员表示，CRISPR介导的DNA胞嘧啶和腺嘌呤碱基编辑工具具有广泛应用，但主要是进行DNA碱基转换（即嘧啶-嘧啶或嘌呤-嘌呤）。

附：英文原文

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

Author: Ibrahim C. Kurt, Ronghao Zhou, Sowmya Iyer, Sara P. Garcia, Bret R. Miller, Lukas M. Langner, Julian Grnewald, J. Keith Joung

Issue&Volume: 2020-07-20

Abstract: CRISPR-guided DNA cytosine and adenine base editors are widely used for many applications1,2,3,4 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.

DOI: 10.1038/s41587-020-0609-x

Source: https://www.nature.com/articles/s41587-020-0609-x

Nature Biotechnology：《自然—生物技术》，创刊于1996年。隶属于施普林格·自然出版集团，最新IF：68.164
官方网址：https://www.nature.com/nbt/
投稿链接：https://mts-nbt.nature.com/cgi-bin/main.plex