Engineered miniature H1 promoters with dedicated RNA polymerase II or III activity

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 h...

Full description

Saved in:
Bibliographic Details
Published inThe Journal of biological chemistry Vol. 296; p. 100026
Main Authors Gao, Zongliang, van der Velden, Yme Ubeles, Fan, Minghui, van der Linden, Cynthia Alyssa, Vink, Monique, Herrera-Carrillo, Elena, Berkhout, Ben
Format Journal Article
LanguageEnglish
Published United States 23.11.2020
Subjects
Pol II
Pol III
RNA polymerase
engineering
H1 promoter
Online AccessGet full text

Cover

More Information
Summary: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).
ISSN:1083-351X