Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
College of Life Science, Tianjin Normal University, Tianjin, China.
Nat Biotechnol. 2021 Jan;39(1):35-40. doi: 10.1038/s41587-020-0592-2. Epub 2020 Jul 20.
Current base editors (BEs) catalyze only base transitions (C to T and A to G) and cannot produce base transversions. Here we present BEs that cause C-to-A transversions in Escherichia coli and C-to-G transversions in mammalian cells. These glycosylase base editors (GBEs) consist of a Cas9 nickase, a cytidine deaminase and a uracil-DNA glycosylase (Ung). Ung excises the U base created by the deaminase, forming an apurinic/apyrimidinic (AP) site that initiates the DNA repair process. In E. coli, we used activation-induced cytidine deaminase (AID) to construct AID-nCas9-Ung and found that it converts C to A with an average editing specificity of 93.8% ± 4.8% and editing efficiency of 87.2% ± 6.9%. For use in mammalian cells, we replaced AID with rat APOBEC1 (APOBEC-nCas9-Ung). We tested APOBEC-nCas9-Ung at 30 endogenous sites, and we observed C-to-G conversions with a high editing specificity at the sixth position of the protospacer between 29.7% and 92.2% and an editing efficiency between 5.3% and 53.0%. APOBEC-nCas9-Ung supplements the current adenine and cytidine BEs (ABE and CBE, respectively) and could be used to target G/C disease-causing mutations.
当前的碱基编辑器（BEs）仅催化碱基转换（C 到 T 和 A 到 G），而不能产生碱基颠换。在这里，我们介绍了能够在大肠杆菌中引起 C 到 A 颠换和在哺乳动物细胞中引起 C 到 G 颠换的碱基编辑器（GBEs）。这些糖基化酶碱基编辑器（GBEs）由 Cas9 切口酶、胞嘧啶脱氨酶和尿嘧啶-DNA 糖基化酶（Ung）组成。Ung 切除脱氨酶产生的 U 碱基，形成无嘌呤/无嘧啶（AP）位点，启动 DNA 修复过程。在大肠杆菌中，我们使用激活诱导胞嘧啶脱氨酶（AID）构建了 AID-nCas9-Ung，并发现它以 93.8%±4.8%的平均编辑特异性和 87.2%±6.9%的编辑效率将 C 转换为 A。为了在哺乳动物细胞中使用，我们用大鼠 APOBEC1（APOBEC-nCas9-Ung）取代了 AID。我们在 30 个内源性位点测试了 APOBEC-nCas9-Ung，观察到在间隔区第六位的 C 到 G 转换具有 29.7%至 92.2%的高编辑特异性和 5.3%至 53.0%的编辑效率。APOBEC-nCas9-Ung 补充了现有的腺嘌呤和胞嘧啶 BEs（分别为 ABE 和 CBE），可用于靶向 G/C 致病突变。
