Double MS2 guided restoration of genetic code in amber (TAG), opal (TGA) and ochre (TAA) stop codon
Double MS2 guided restoration of genetic code in amber (TAG), opal (TGA) and ochre (TAA) stop codon
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DOI：
10.1016/j.enzmictec.2021.109851
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发表时间：
2021-06-17
影响因子：
3.4
通讯作者：
Tsukahara, Toshifumi
中科院分区：
文献类型：
--
作者：
Bhakta, Sonali;Tsukahara, Toshifumi
基因治疗对常见遗传病的普及和前景目前正在增加。虽然对遗传性疾病的有效治疗是罕见的，但编辑突变基因是由终止密码子突变引起的疾病的可能治疗方法，包括琥珀（TAG），蛋白石（TGA）或赭石（TAA）终止密码子。使用人工RNA编辑的点突变RNA的恢复可用于修改基因编码的信息并从单个基因产生功能上不同的蛋白质。通过将RNA编辑酶（作用于RNA的腺苷脱氨酶（阿达尔））的催化结构域连接至反义引导RNA，特异性腺苷（A）可转化为肌苷（I），其在翻译期间被识别为鸟苷（G）。在这项研究中，我们设计了ADAR 1和MS 2系统的脱氨酶结构域，以靶向特定的腺苷并恢复G至A突变。为此，ADAR 1脱氨酶结构域与RNA结合蛋白MS 2融合，MS 2与MS 2 RNA结合。将19 bp的引导RNA设计为与靶mRNA互补，在引导RNA下游具有6X茎环和CMV启动子，或在引导RNA的任一侧具有1X MS 2茎环和U6启动子。工程化的ADAR 1脱氨酶结构域可以在EGFP中所需的编辑位点将腺苷转化为肌苷，所述编辑位点被编辑为包含琥珀（TAG）、蛋白石（TGA）或赭石（TAA）终止密码子。该系统可以在细胞系统中将终止密码子转换为通读色氨酸密码子（TGG），从而导致使用JuLi显微镜观察到的荧光发射。还进行了靶转录物的PCR-RFLP和桑格测序，揭示了使用双MS 2对蛋白石终止密码子的编辑效率为20.97%，对赭石终止密码子中的5 '和3 ' A残基的编辑效率分别为26%和17%。这比使用MS 2 -6X向导RNA实现的编辑率更高。随时间从三种不同终止密码子恢复通读密码子的观察表明，24小时后编辑密码子的百分比相对较低，48小时后增加，但72小时后再次下降。这一系统的成功建立有可能代表基因治疗领域的一个新时代。
The popularity and promise of gene therapy for common genetic diseases are currently increasing. Although effective treatments for genetic disorders are rare, editing of the mutated gene is a possible therapeutic approach for conditions caused by stop codon mutations, including either amber (TAG), opal (TGA) or ochre (TAA) stop codons. Restoration of point-mutated RNAs using artificial RNA editing can be used to modify gene-encoded information and generate functionally distinct proteins from a single gene. By linking the catalytic domain of the RNA editing enzyme, adenosine deaminase acting on RNA (ADAR), to an antisense guide RNA, specific adenosines (A) can be converted to inosine (I), which is recognized as guanosine (G) during translation. In this study, we engineered the deaminase domain of ADAR1 and the MS2 system to target a specific adenosine and restore the G to A mutations. To this end, the ADAR1 deaminase domain was fused with the RNA binding protein, MS2, which binds to MS2 RNA. Guide RNAs of 19 bp were designed to be complementary to target mRNAs, with either 6X stem-loops downstream of the guide RNA and a CMV promoter, or a 1X MS2 stem-loop on either side of the guide RNA and a U6 promoter. The engineered ADAR1 deaminase domain could convert adenosine to inosine at the desired editing site in EGFP, which was edited to contain an amber (TAG), opal (TGA) or ochre (TAA) stop codon. The system could convert the stop codons to a read-through tryptophan codon (TGG) in a cellular system, leading to fluorescence emission, observed using JuLi microscopy. PCR-RFLP and Sanger sequencing of the target transcript were also conducted, revealing an editing efficiency of 20.97 % for the opal stop codon, and 26 % and 17 % for the 5 ' and 3 ' A residues, respectively, in the ochre stop codon, using the double MS2. This was a higher editing rate than that achieved using the MS2-6X guide RNA. Observation of restoration of the read-through codon from the three different stop codons over time demonstrated a relatively low percentage of edited codons after 24 h, which increased after 48 h, but decreased again after 72 h. Successful establishment of this system has the potential to represent a new era in the field of gene therapy.