School of Life Sciences, Shanghai University, Shanghai, 200444, China.
Key Laboratory of Molecular Virology and Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, 200031, China.
ACS Synth Biol. 2021 May 21;10(5):1106-1115. doi: 10.1021/acssynbio.0c00627. Epub 2021 May 2.
APOBEC3A (A3A) is a cytidine deaminase involved in innate immune response and is able to catalyze deamination on both DNA and RNA substrates. It was used in creating the CRISPR-mediated base editor, but has since been held back due to its dual activities. On the other hand, it has been a challenge to separate A3A's dual activities in order to enable it for single-base RNA editors. Here we developed the reporter system for C-to-U RNA editing and employed rational design for mutagenesis to differentiate deaminase activities on RNA and DNA substrates to obtain an RNA-specific editase. Generation and examination of 23 previous A3A mutants showed their deamination activity on RNA was mostly abolished when their activity on DNA was impaired, with the exception of mutant N57Q that displayed an inverse change. We designed new mutations on Loops 1 and 7 based on A3A's crystal structure and found mutants H29R and Y132G had differential effects on catalytic activity on RNA and DNA substrates. In order to engineer an A3A with RNA-specific deaminase activity, we combined Y132G with mutations in Loop 1 or helix 6 by rational design. Two multipoint mutants, Y132G/K30R and Y132G/G188A/R189A/L190A, were successful in retaining high deaminase activity on RNA substrate while eliminating deaminase activity on DNA. We, for the first time, created novel human A3A variants with RNA-specific cytidine deaminase activity, providing insight into A3A's mechanism on substrate recognition and a new addition of a toolset to the creation of a RNA-specific C-to-U base editor.
APOBEC3A（A3A）是一种参与先天免疫反应的胞嘧啶脱氨酶，能够在 DNA 和 RNA 底物上催化脱氨作用。它被用于创建 CRISPR 介导的碱基编辑器，但由于其双重活性而受到限制。另一方面，分离 A3A 的双重活性以使其能够用于单碱基 RNA 编辑器一直是一个挑战。在这里，我们开发了用于 C 到 U RNA 编辑的报告系统，并采用合理的设计进行诱变，以区分 RNA 和 DNA 底物上的脱氨酶活性，从而获得 RNA 特异性编辑酶。对 23 个先前的 A3A 突变体的生成和检查表明，当它们在 DNA 上的活性受损时，它们在 RNA 上的脱氨活性大多被消除，除了突变体 N57Q 显示出相反的变化。我们根据 A3A 的晶体结构在环 1 和 7 上设计了新的突变，并发现突变体 H29R 和 Y132G 对 RNA 和 DNA 底物的催化活性有不同的影响。为了设计具有 RNA 特异性脱氨酶活性的 A3A，我们通过合理设计将 Y132G 与环 1 或螺旋 6 中的突变结合使用。两个多点突变体，Y132G/K30R 和 Y132G/G188A/R189A/L190A，成功地在保留对 RNA 底物的高脱氨酶活性的同时消除了对 DNA 的脱氨酶活性。我们首次创建了具有 RNA 特异性胞嘧啶脱氨酶活性的新型人类 A3A 变体，为 A3A 对底物识别的机制提供了深入了解，并为创建 RNA 特异性 C 到 U 碱基编辑器提供了新的工具集。
