RNA editing of BFP, a point mutant of GFP, using artificial APOBEC1 deaminase to restore the genetic code.
RNA editing of BFP, a point mutant of GFP, using artificial APOBEC1 deaminase to restore the genetic code.
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利用人工载脂蛋白B mRNA编辑酶催化多肽1（APOBEC1）脱氨酶对绿色荧光蛋白（GFP）的点突变体蓝色荧光蛋白（BFP）进行RNA编辑，以恢复遗传密码 。
DOI：
10.1038/s41598-020-74374-5
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发表时间：
2020-10-14
影响因子：
4.6
通讯作者：
Tsukahara T
中科院分区：
文献类型：
--
作者：
Bhakta S;Sakari M;Tsukahara T
许多遗传性疾病是由T到C的点突变引起的。因此，对突变基因进行编辑是治疗这些疾病的一种有前景的策略。我们通过将载脂蛋白B mRNA编辑催化多肽1（APOBEC1）的脱氨酶结构域与一种与目标mRNA互补的向导RNA（gRNA）相结合，构建了一种人工RNA编辑酶。在这个人工酶系统中，gRNA与MS2茎环结构结合，而具有将突变的目标核苷酸C转变为U能力的脱氨酶结构域则与MS2外壳蛋白融合。我们使用编码蓝色荧光蛋白（BFP）的RNA作为目标RNA，该RNA由编码绿色荧光蛋白（GFP）的基因经199位T＞C突变而来。在这两个组件（脱氨酶和gRNA）瞬时表达后，我们通过共聚焦显微镜观察到了绿色荧光蛋白，这表明BFP中突变的199位C已转变为U，恢复了GFP的原始序列。这一结果通过对转染细胞的cDNA进行聚合酶链反应-限制性片段长度多态性分析（PCR–RFLP）和桑格测序得到了证实，结果显示编辑效率约为21%。尽管深度RNA测序结果显示该系统中存在一些脱靶编辑事件，但我们成功开发出了一种利用人工脱氨酶（APOBEC1）结合MS2系统的人工RNA编辑系统，这可能会带来通过在mRNA水平恢复野生型序列来治疗遗传性疾病的疗法 。
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.
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影响因子：
64.8
作者：
Komor AC;Kim YB;Packer MS;Zuris JA;Liu DR
通讯作者：
Liu DR
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
影响因子：
4.5
作者：
Chester, A;Weinreb, V;Navaratnam, N
通讯作者：
Navaratnam, N
影响因子：
2.4
作者：
Bhakta, Sonali;Azad, Md Thoufic Anam;Tsukahara, Toshifumi
通讯作者：
Tsukahara, Toshifumi
影响因子：
5.1
作者：
Azad, Md T. A.;Bhakta, S.;Tsukahara, T.
通讯作者：
Tsukahara, T.