Area of Bioscience, Biotechnology and Biomedical Engineering Research Area, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City 923-1292, Ishikawa, Japan.
Department of Veterinary and Animal Sciences, University of Rajshahi, Rajshahi 6205, Bangladesh.
Genes (Basel). 2023 Aug 4;14(8):1584. doi: 10.3390/genes14081584.
Adenosine deaminases acting on RNA (ADARs) have double-stranded RNA binding domains and a deaminase domain (DD). We used the MS2 system and specific guide RNAs to direct ADAR1-DD to target adenosines in the mRNA encoding-enhanced green fluorescence protein. Using this system in transfected HEK-293 cells, we evaluated the effects of changing the length and position of the guide RNA on the efficiency of conversion of amber (TAG) and ochre (TAA) stop codons to tryptophan (TGG) in the target. Guide RNAs of 19, 21 and 23 nt were positioned upstream and downstream of the MS2-RNA, providing a total of six guide RNAs. The upstream guide RNAs were more functionally effective than the downstream guide RNAs, with the following hierarchy of efficiency: 21 nt > 23 nt > 19 nt. The highest editing efficiency was 16.6%. Off-target editing was not detected in the guide RNA complementary region but was detected 50 nt downstream of the target. The editing efficiency was proportional to the amount of transfected deaminase but inversely proportional to the amount of the transfected guide RNA. Our results suggest that specific RNA editing requires precise optimization of the ratio of enzyme, guide RNA, and target RNA.
腺苷脱氨酶作用于 RNA（ADARs）具有双链 RNA 结合域和脱氨酶域（DD）。我们使用 MS2 系统和特定的指导 RNA 引导 ADAR1-DD 靶向编码增强型绿色荧光蛋白的 mRNA 中的腺苷。使用该系统在转染的 HEK-293 细胞中，我们评估了改变指导 RNA 的长度和位置对目标中琥珀（TAG）和赭石（TAA）终止密码子转换为色氨酸（TGG）的效率的影响。19、21 和 23 个核苷酸的指导 RNA 位于 MS2-RNA 的上下游，总共提供了 6 个指导 RNA。上游指导 RNA 比下游指导 RNA 更有效，效率顺序如下：21 nt > 23 nt > 19 nt。最高编辑效率为 16.6%。在指导 RNA 互补区域未检测到脱靶编辑，但在靶标 50 个核苷酸下游检测到脱靶编辑。编辑效率与转染的脱氨酶量成正比，但与转染的指导 RNA 量成反比。我们的结果表明，特定的 RNA 编辑需要精确优化酶、指导 RNA 和靶 RNA 的比例。
