In eukaryotes, the CRISPR-Cas9 system has now been widely used as a revolutionary genome engineering tool^{1, 2}. However, in prokaryotes, the use of nuclease-mediated genome editing tools has been limited to negative selection for the already modified cells because of its lethality^{3, 4}. Here, we report on deaminase-mediated targeted nucleotide editing (Target-AID) ⁵ adopted in Escherichia coli. Cytidine deaminase PmCDA1 fused to the nuclease-deficient CRISPR-Cas9 system achieved specific point mutagenesis at the target sites in E. coli by introducing cytosine mutations without compromising cell growth. The cytosine-to-thymine substitutions were induced mainly within an approximately five-base window of target sequences on the protospacer adjacent motif-distal side, which can be shifted depending on the length of the single guide RNA sequence. Use of a uracil DNA glycosylase inhibitor ⁶ in combination with a degradation tag (LVA tag) ⁷ resulted in a robustly high mutation efficiency, which allowed simultaneous multiplex editing of six different genes. The major multi-copy transposase genes that consist of at least 41 loci were also simultaneously edited by using four target sequences. As this system does not rely on any additional or host-dependent factors, it may be readily applicable to a wide range of bacteria.

中文翻译：

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大肠杆菌中脱氨酶介导的多重基因组编辑。

在真核生物中，CRISPR-Cas9系统现已被广泛用作革命性的基因组工程工具^1、2。然而，在原核生物中，由于其致死性^3，核酸酶介导的基因组编辑工具的使用仅限于对已经修饰的细胞进行阴性选择。在这里，我们报道脱氨酶介导的靶向核苷酸编辑（Target-AID）⁵在大肠杆菌中被采用。融合至核酸酶缺陷型CRISPR-Cas9系统的胞苷脱氨酶PmCDA1通过引入胞嘧啶突变而不会损害细胞生长，从而在大肠杆菌的靶位点实现了特异性点诱变。胞嘧啶至胸腺嘧啶的取代主要是在原间隔物邻近基序-远端侧的靶序列的大约五碱基窗口内诱导的，其可以根据单个指导RNA序列的长度而移动。尿嘧啶DNA糖基化酶抑制剂⁶与降解标签（LVA标签）结合使用⁷导致了极高的突变效率，可以同时对六个不同的基因进行多重编辑。还通过使用四个靶序列同时编辑了由至少41个基因座组成的主要多拷贝转座酶基因。由于该系统不依赖于任何其他或依赖宿主的因素，因此可以很容易地应用于各种细菌。