Site directed mutagenesis is a very effective approach to recode genetic information. Proper linking of the catalytic domain of the RNA editing enzyme adenosine deaminase acting on RNA (ADAR) to an antisense guide RNA can convert specific adenosines (As) to inosines (Is), with the latter recognized as guanosines (Gs) during the translation process. Efforts have been made to engineer the deaminase domain of ADAR1 and the MS2 system to target specific A residues to restore G→A mutations. The target consisted of an ochre (TAA) stop codon, generated from the TGG codon encoding amino acid 58 (Trp) of enhanced green fluorescent protein (EGFP). This system had the ability to convert the stop codon (TAA) to a readable codon (TGG), thereby restoring fluorescence in a cellular system, as shown by JuLi fluorescence and LSM confocal microscopy. The specificity of the editing was confirmed by polymerase chain reaction-restriction fragment length polymorphism, as the restored EGFP mRNA could be cleaved into fragments of 160 and 100 base pairs. Direct sequencing analysis with both sense and antisense primers showed that the restoration rate was higher for the 5′ than for the 3′A. This system may be very useful for treating genetic diseases that result from G→A point mutations. Successful artificial editing of RNA in vivo can accelerate research in this field, and pioneer genetic code restoration therapy, including stop codon read-through therapy, for various genetic diseases.

中文翻译：

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通过ADAR1脱氨酶对RNA石终止密码子进行人工RNA编辑来恢复遗传密码

定点诱变是一种非常有效的方法来重新编码遗传信息。RNA编辑酶作用于RNA（ADAR）的腺苷脱氨酶催化域与反义向导的正确连接可以将特定的腺苷（As）转化为肌苷（Is），后者在翻译过程中被称为鸟苷（Gs） 。已经努力改造ADAR1和MS2系统的脱氨酶结构域以靶向特定的A残基以恢复G→A突变。该靶标由an石（TAA）终止密码子组成，其由编码增强型绿色荧光蛋白（EGFP）的氨基酸58（Trp）的TGG密码子产生。该系统具有将终止密码子（TAA）转换为可读密码子（TGG）的能力，从而可以在细胞系统中恢复荧光，如JuLi荧光和LSM共聚焦显微镜所示。通过聚合酶链反应-限制性片段长度多态性证实了编辑的特异性，因为恢复的EGFP mRNA可以切割成160和100个碱基对的片段。使用有义和反义引物的直接测序分析表明，5'的恢复率高于3'A。该系统对于治疗由G→A点突变引起的遗传疾病可能非常有用。成功的体内RNA人工编辑可以加速该领域的研究，并开创了用于各种遗传疾病的遗传密码恢复疗法，包括终止密码子通读疗法。使用有义和反义引物的直接测序分析表明，5'的恢复率高于3'A。该系统对于治疗由G→A点突变引起的遗传疾病可能非常有用。成功的体内RNA人工编辑可以加速该领域的研究，并开创了用于各种遗传疾病的遗传密码恢复疗法，包括终止密码子通读疗法。使用有义和反义引物的直接测序分析表明，5'的恢复率高于3'A。该系统对于治疗由G→A点突变引起的遗传疾病可能非常有用。成功的体内RNA人工编辑可以加速该领域的研究，并开创了用于各种遗传疾病的遗传密码恢复疗法，包括终止密码子通读疗法。