Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions.
Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions.
复制标题
DOI：
10.1038/nbt.3803
复制
发表时间：
2017-04
影响因子：
46.9
通讯作者：
Liu DR
中科院分区：
文献类型：
--
作者：
Kim YB;Komor AC;Levy JM;Packer MS;Zhao KT;Liu DR
碱基编辑是最近开发的基因组编辑方法，其使用含有催化缺陷的化脓链球菌Cas9、胞苷脱氨酶和碱基切除修复抑制剂的融合蛋白来诱导活细胞DNA中的可编程的单核苷酸变化，而不产生双链DNA断裂，不需要供体DNA模板，并且不诱导过量的随机插入和缺失。在这里，我们报告了五种新的C→T（或G→A）碱基编辑器的开发，这些编辑器使用具有不同前间区序列邻近基序（PAM）特异性的天然和工程化Cas9变体，以将可以通过碱基编辑靶向的位点数量扩大2.5倍。此外，我们设计了含有突变的胞苷脱氨酶结构域的新碱基编辑器，其将表观编辑窗口的宽度从约5个核苷酸缩小到少至1至2个核苷酸，使得能够区分先前以相当的效率编辑的相邻C核苷酸，从而使可以优先校正的疾病相关靶C的数量加倍超过附近的非靶C。总的来说，这些发展大大增加了基础编辑的目标范围，并建立了基础编辑器的模块化性质。
Base editing is a recently developed approach to genome editing that uses a fusion protein containing a catalytically defective Streptococcus pyogenes Cas9, a cytidine deaminase, and an inhibitor of base excision repair to induce programmable, single-nucleotide changes in the DNA of living cells without generating double-strand DNA breaks, without requiring a donor DNA template, and without inducing an excess of stochastic insertions and deletions. Here we report the development of five new C→T (or G→A) base editors that use natural and engineered Cas9 variants with different protospacer-adjacent motif (PAM) specificities to expand the number of sites that can be targeted by base editing by 2.5-fold. Additionally, we engineered new base editors containing mutated cytidine deaminase domains that narrow the width of the apparent editing window from approximately 5 nucleotides to as little as 1 to 2 nucleotides, enabling the discrimination of neighboring C nucleotides that would previously be edited with comparable efficiency, thereby doubling the number of disease-associated target Cs that can be corrected preferentially over nearby non-target Cs. Collectively, these developments substantially increase the targeting scope of base editing and establish the modular nature of base editors.