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：

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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。