小柯机器人

低脱靶效率碱基编辑器的发现
2020-02-13 10:09

美国博德研究所David R. Liu研究小组近日取得一项新成果。他们通过评估胞嘧啶碱基编辑器 (CBE),发现最小化Cas9非依赖的脱靶DNA编辑器。2020年2月10日,国际学术期刊《自然—生物技术》在线发表了这一成果。

研究人员利用多种快速、经济高效的方法来筛选不同CBE在大肠杆菌和人细胞中诱导Cas9非依赖的脱氨倾向。研究人员使用这些测定方法来筛选具有低Cas9非依赖脱氨基作用的CBE,并通过全基因组测序发现YE1(CBE变体)具有Cas9非依赖脱靶编辑的基本水平。研究人员设计了YE1变异体,该变异体保留了CBE靶向底物的高活性,同时保持了最低的Cas9非依赖的脱靶编辑。该研究鉴定和设计的CBE套件具约有低于平均值10-100倍的Cas9非依赖脱靶编辑效率,同时具有典型CBE可以靶向大多数位置的稳定编辑特性,因此特别适合在需要最小化脱靶编辑效率的实验中应用。

据介绍,CBE使基因组DNA中CG碱基转化为TA碱基。最近的研究显示,原始的CBE BE3在小鼠胚胎和水稻的全基因组中诱导了低频率且Cas9非依赖的脱靶CG-TA突变。

附:英文原文

Title: Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors

Author: Jordan L. Doman, Aditya Raguram, Gregory A. Newby, David R. Liu

Issue&Volume: 2020-02-10

Abstract: Cytosine base editors (CBEs) enable targeted CG-to-TA conversions in genomic DNA. Recent studies report that BE3, the original CBE, induces a low frequency of genome-wide Cas9-independent off-target CG-to-TA mutation in mouse embryos and in rice. Here we develop multiple rapid, cost-effective methods to screen the propensity of different CBEs to induce Cas9-independent deamination in Escherichia coli and in human cells. We use these assays to identify CBEs with reduced Cas9-independent deamination and validate via whole-genome sequencing that YE1, a narrowed-window CBE variant, displays background levels of Cas9-independent off-target editing. We engineered YE1 variants that retain the substrate-targeting scope of high-activity CBEs while maintaining minimal Cas9-independent off-target editing. The suite of CBEs characterized and engineered in this study collectively offer ~10–100-fold lower average Cas9-independent off-target DNA editing while maintaining robust on-target editing at most positions targetable by canonical CBEs, and thus are especially promising for applications in which off-target editing must be minimized.

DOI: 10.1038/s41587-020-0414-6

Source: https://www.nature.com/articles/s41587-020-0414-6

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


本期文章:《自然—生物技术》:Online/在线发表

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