Engineered miniature H1 promoters with dedicated RNA polymerase II or III activity.
Engineered miniature H1 promoters with dedicated RNA polymerase II or III activity.
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DOI：
10.1074/jbc.ra120.015386
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发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Berkhout B
中科院分区：
文献类型：
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作者：
Gao Z;van der Velden YU;Fan M;van der Linden CA;Vink M;Herrera-Carrillo E;Berkhout B
RNA聚合酶III（Pol III）启动子，如7SK、U6和H1，广泛用于表达小的非编码RNA，包括用于RNAi实验的短发夹RNA和用于CRISPR介导的基因组编辑的指导RNA。我们以前报告的双重RNA聚合酶活性（Pol II/III）的人H1启动子，并证明，这种混杂的RNA聚合酶的使用可以利用同时表达的非编码RNA和mRNA。然而，这种组合在其他实验和治疗环境中不是期望的特征。为了克服H1启动子的这一限制，我们设计了具有最小Pol II活性的微型H1/7SK杂合启动子，从而将Pol III活性提高到高于任一亲本启动子的水平。同时，我们还设计了小的Pol II特异性H1启动子变体，并探索了它们作为蛋白质表达的通用Pol II启动子的用途。新工程化的启动子变体不仅在活性和小启动子大小方面，而且在通过所需治疗转录物（pol II或pol III，但不是两者）的排他性表达而涉及安全性方面，形成了常用H1启动子的有吸引力的替代物。
RNA polymerase III (Pol III) promoters, such as 7SK, U6, and H1, are widely used for the expression of small noncoding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a noncoding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter, we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly used H1 promoter in terms of not only activity and small promoter size but also concerning safety by exclusive expression of the desired therapeutic transcript (either pol II or pol III but not both).