Adenosine deaminases acting on RNA (ADARs) can be repurposed to enable programmable RNA editing, however their exogenous delivery leads to transcriptome-wide off-targeting, and additionally, enzymatic activity on certain RNA motifs, especially those flanked by a 5′ guanosine is very low thus limiting their utility as a transcriptome engineering toolset. To address this, we explored comprehensive ADAR2 protein engineering via three approaches: First, we performed a novel deep mutational scan of the deaminase domain that enabled direct coupling of variants to corresponding RNA editing activity. Experimentally measuring the impact of every amino acid substitution across 261 residues, i.e. ~5000 variants, on RNA editing, revealed intrinsic domain properties, and also several mutations that greatly enhanced RNA editing. Second, we performed a domain-wide mutagenesis screen to identify variants that increased activity at 5′-GA-3′ motifs, and discovered novel mutants that enabled robust RNA editing. Third, we engineered the domain at the fragment level to create split deaminases. Notably, compared to full-length deaminase overexpression, split-deaminases resulted in >1000 fold more specific RNA editing. Taken together, we anticipate this comprehensive deaminase engineering will enable broader utility of the ADAR toolset for RNA biotechnology and therapeutic applications.

中文翻译：

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全面询问ADAR2脱氨酶结构域，以工程设计增强的RNA碱基编辑活性，功能和特异性

可以将作用于RNA（ADAR）的腺苷脱氨酶重新用于实现可编程RNA的编辑，但是它们的外源传递导致转录组范围内的脱靶，此外，对某些RNA序列，尤其是那些侧接5'鸟苷的RNA的酶促活性非常高。低，因此限制了它们作为转录组工程工具集的效用。为了解决这个问题，我们通过三种方法探索了全面的ADAR2蛋白工程：首先，我们对脱氨酶域进行了新颖的深层突变扫描，该扫描使变体与相应的RNA编辑活性直接偶联。通过实验测量跨越261个残基（即约5000个变体）的每个氨基酸取代对RNA编辑的影响，揭示了固有的域结构特性，并且还发现了几个大大增强RNA编辑的突变。第二，我们进行了全域诱变筛选，以鉴定可提高5'-GA-3'基序活性的变体，并发现了能够进行强大RNA编辑的新型突变体。第三，我们在片段级别设计域以创建拆分的脱氨基。值得注意的是，与全长脱氨酶的过表达相比，分裂脱氨酶的特异RNA编辑具有> 1000倍的特异性。综上所述，我们期望这种全面的脱氨酶工程技术将使ADAR工具集在RNA生物技术和治疗应用中具有更广泛的用途。分开的脱氨基酶导致特异的RNA编辑> 1000倍。综上所述，我们期望这种全面的脱氨酶工程技术将使ADAR工具集在RNA生物技术和治疗应用中具有更广泛的用途。分开的脱氨基酶导致特异的RNA编辑> 1000倍。综上所述，我们期望这种全面的脱氨酶工程技术将使ADAR工具集在RNA生物技术和治疗应用中具有更广泛的用途。