期刊
MOLECULAR THERAPY-NUCLEIC ACIDS
卷 10, 期 -, 页码 36-44出版社
CELL PRESS
DOI: 10.1016/j.omtn.2017.11.006
关键词
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资金
- NWO-Chemical Sciences (TOP grant)
- ZonMW
- China Scholarship Council (CSC)
Type 3 Pol III promoters such as U6 are widely used for expression of small RNAs, including short hairpin RNA for RNAi applications and guide RNA in CRISPR genome-editing platforms. RNA polymerase III uses a T-stretch as termination signal, but the exact properties have not been thoroughly investigated. Here, we systematically measured the in vivo termination efficiency and the actual site of termination for different T-stretch signals in three commonly used human Pol III promoters (U6, 7SK, and H1). Both the termination efficiency and the actual termination site depend on the T-stretch signal. The T4 signal acts as minimal terminator, but full termination efficiency is reached only with a T-stretch of >= 6. The termination site within the T-stretch is quite heterogeneous, and consequently small RNAs have a variable U-tail of 1-6 nucleotides. We further report that such variable U-tails can have a significant negative effect on the functionality of the crRNA effector of the CRISPR-AsCpf1 system. We next improved these crRNAs by insertion of the HDV ribozyme to avoid U-tails. This study provides detailed design guidelines for small RNA expression cassettes based on Pol III.
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