Department of Bioengineering, University of California San Diego, San Diego, United States.
Division of Biological Sciences, University of California San Diego, San Diego, United States.
Elife. 2022 Jan 19;11:e75555. doi: 10.7554/eLife.75555.
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. Towards addressing these issues, we first performed a novel deep mutational scan of the ADAR2 deaminase domain, directly measuring the impact of every amino acid substitution across 261 residues, on RNA editing. This enabled us to create a domain-wide mutagenesis map while also revealing a novel hyperactive variant with improved enzymatic activity at ' motifs. As overexpression of ADAR enzymes, especially hyperactive variants, can lead to significant transcriptome-wide off-targeting, we next engineered a split-ADAR2 deaminase which resulted in >100-fold more specific RNA editing as compared to full-length deaminase overexpression. Taken together, we anticipate this systematic engineering of the ADAR2 deaminase domain will enable broader utility of the ADAR toolset for RNA biotechnology applications.
腺苷脱氨酶作用于 RNA（ADARs）可被重新用于实现可编程 RNA 编辑，然而，它们的外源性递送会导致全转录组范围的脱靶效应，此外，某些 RNA 基序（特别是侧翼为 5'鸟苷的基序）上的酶活性非常低，从而限制了它们作为转录组工程工具集的用途。为了解决这些问题，我们首先对 ADAR2 脱氨酶结构域进行了新颖的深度突变扫描，直接测量了 261 个残基上每个氨基酸取代对 RNA 编辑的影响。这使我们能够创建一个全域诱变图谱，同时还揭示了一种新型的超活性变体，其在“motifs”上具有提高的酶活性。由于 ADAR 酶的过表达，特别是超活性变体的过表达，可能导致全转录组范围的脱靶效应，因此我们接下来设计了一种分割 ADAR2 脱氨酶，与全长脱氨酶过表达相比，其导致的 RNA 编辑特异性提高了 100 多倍。总之，我们预计对 ADAR2 脱氨酶结构域的这种系统工程改造将使 ADAR 工具集更广泛地用于 RNA 生物技术应用。
