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Efficient Gene Silencing by Adenine Base Editor-Mediated Start Codon Mutation.
Molecular Therapy ( IF 12.1 ) Pub Date : 2019-11-29 , DOI: 10.1016/j.ymthe.2019.11.022
Xinjie Wang 1 , Zhiwei Liu 2 , GuangLei Li 3 , Lu Dang 4 , Shisheng Huang 3 , Lei He 2 , Yu'e Ma 2 , Cong Li 2 , Ming Liu 5 , Guang Yang 3 , Xingxu Huang 3 , Fei Zhou 2 , Xiaodong Ma 1
Affiliation  

Traditional CRISPR/Cas9-based gene knockouts are generated by introducing DNA double-strand breaks (DSBs), but this may cause excessive DNA damage or cell death. CRISPR-based cytosine base editors (CBEs) and adenine base editors (ABEs) can facilitate C-to-T or A-to-G exchanges, respectively, without DSBs. CBEs have been employed in a gene knockout strategy: CRISPR-STOP or i-STOP changes single nucleotides to induce in-frame stop codons. However, this strategy is not applicable for some genes, and the unwanted mutations in CBE systems have recently been reported to be surprisingly significant. As a variant, the ABE systems mediate precise editing and have only rare unwanted mutations. In this study, we implemented a new strategy to induce gene silencing (i-Silence) with an ABE-mediated start codon mutation from ATG to GTG or ACG. Using both in vitro and in vivo model systems, we showed that the i-Silence approach is efficient and precise for producing a gene knockout. In addition, the i-Silence strategy can be employed to analyze ~17,804 human genes and can be used to mimic 147 kinds of pathogenic diseases caused by start codon mutations. Altogether, compared to other methods, the ABE-based i-Silence method is a safer gene knockout strategy, and it has promising application potential.

中文翻译:

腺嘌呤碱基编辑介导的起始密码子突变可实现有效的基因沉默。

传统的基于CRISPR / Cas9的基因敲除是通过引入DNA双链断裂(DSB)产生的,但这可能会导致DNA过度损伤或细胞死亡。基于CRISPR的胞嘧啶碱基编辑器(CBE)和腺嘌呤碱基编辑器(ABE)可以分别促进C-to-T或A-to-G交换,而无需DSB。CBE已用于基因敲除策略:CRISPR-STOP或i-STOP改变单个核苷酸以诱导框内终止密码子。但是,该策略不适用于某些基因,并且最近有报道称CBE系统中的不需要的突变具有惊人的意义。作为一种变体,ABE系统可以进行精确的编辑,并且只有很少的不需要的突变。在这项研究中,我们实施了一种新的策略来诱导ABE介导的起始密码子突变(从ATG到GTG或ACG)的基因沉默(i-Silence)。通过使用体外和体内模型系统,我们证明了i-Silence方法对于产生基因敲除是有效而精确的。此外,i-Silence策略可用于分析〜17,804个人类基因,并可用于模拟由起始密码子突变引起的147种致病性疾病。总之,与其他方法相比,基于ABE的i-Silence方法是一种更安全的基因敲除策略,具有广阔的应用前景。
更新日期:2019-11-29
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