Area of Bioscience and Biotechnology, School of Materials Science, Japan Advanced Institute of Science and Technology, M1-4F, 1-1 Asahidai, Nomi City, Ishikawa, 923-1292, Japan.
Sci Rep. 2020 Oct 14;10(1):17304. doi: 10.1038/s41598-020-74374-5.
Many genetic diseases are caused by T-to-C point mutations. Hence, editing of mutated genes represents a promising strategy for treating these disorders. We engineered an artificial RNA editase by combining the deaminase domain of APOBEC1 (apolipoprotein B mRNA editing catalytic polypeptide 1) with a guideRNA (gRNA) which is complementary to target mRNA. In this artificial enzyme system, gRNA is bound to MS2 stem-loop, and deaminase domain, which has the ability to convert mutated target nucleotide C-to-U, is fused to MS2 coat protein. As a target RNA, we used RNA encoding blue fluorescent protein (BFP) which was derived from the gene encoding GFP by 199 T > C mutation. Upon transient expression of both components (deaminase and gRNA), we observed GFP by confocal microscopy, indicating that mutated 199C in BFP had been converted to U, restoring original sequence of GFP. This result was confirmed by PCR-RFLP and Sanger's sequencing using cDNA from transfected cells, revealing an editing efficiency of approximately 21%. Although deep RNA sequencing result showed some off-target editing events in this system, we successfully developed an artificial RNA editing system using artificial deaminase (APOBEC1) in combination with MS2 system could lead to therapies that treat genetic disease by restoring wild-type sequence at the mRNA level.
许多遗传疾病是由 T 到 C 的点突变引起的。因此，编辑突变基因代表了治疗这些疾病的一种有前途的策略。我们通过将 APOBEC1（载脂蛋白 B mRNA 编辑酶催化多肽 1）的脱氨酶结构域与互补于靶 mRNA 的向导 RNA（gRNA）结合，设计了一种人工 RNA 编辑酶。在这个人工酶系统中，gRNA 与 MS2 茎环结合，而具有将突变靶核苷酸 C 转换为 U 的能力的脱氨酶结构域融合到 MS2 衣壳蛋白上。作为靶 RNA，我们使用 RNA 编码蓝色荧光蛋白（BFP），该 RNA 是通过 GFP 基因中的 199T>C 突变衍生而来的。在瞬时表达两种成分（脱氨酶和 gRNA）后，我们通过共聚焦显微镜观察到 GFP，表明 BFP 中突变的 199C 已被转换为 U，恢复了 GFP 的原始序列。这一结果通过从转染细胞中提取的 cDNA 进行 PCR-RFLP 和 Sanger 测序得到了证实，显示编辑效率约为 21%。尽管深度 RNA 测序结果表明该系统存在一些脱靶编辑事件，但我们成功开发了一种使用人工脱氨酶（APOBEC1）与 MS2 系统相结合的人工 RNA 编辑系统，该系统可能会通过在 mRNA 水平上恢复野生型序列来治疗遗传疾病。
