Efficacy, accumulation, and transcriptional profile of anti-HIV shRNAs expressed from human U6, 7SK, and H1 promoters

Abstract

The expression of short hairpin RNAs (shRNAs) in cells has many potential therapeutic applications, including as a functional cure for HIV. The RNA polymerase III promoters H1, 7SK, and U6 have all been used to express shRNAs. However, there have been no direct and simultaneous comparisons of shRNA potency, expression level, and transcriptional profile between the promoters. We show that the 7SK and U6 promoters result in higher shRNA levels and potency compared to the H1 promoter but that in transduced T lymphocytes, higher expression levels can also lead to growth defects. We present evidence that Dicer cleavage of shRNAs is measured from the first base pair in the shRNA stem, rather than from the 5 0 end as previously shown for structurally related microRNAs. As a result, guide-strand identity was unaffected by variations in 5 0 transcription start sites among the different promoters, making expression levels the main determinant of shRNA potency. While all promoters generated shRNAs with variable start sites, the U6 promoter was the most accurate in using its intended +1 position. Our results have implications for the development of therapeutic small RNAs for gene therapy and for our understanding of how shRNAs are processed in cells.

A versatile, high-efficiency platform for CRISPR-based gene activation

“…Manipulation of the SA guide RNA and/or its expression may provide yet another avenue for future optimization. In our system, the SA guide expression context (H1 promoter combined with the 2.0 sgRNA scaffold) is suboptimal relative to the higher-efficiency U6 promoter and GNE-3 sgRNA employed for gene-targeting guides 30 . This may enable selection of more active CRISPRa populations, as only those cells which can activate the Puro r gene under such limiting conditions will survive.…”

Harnessing Catalytic RNA Circuits for Construction of Artificial Signaling Pathways in Mammalian Cells

“…We firstly chose the Luciferase mRNA as the trigger to initiate CHA (Figure S1) and target another endogenous gene via CRISPR‐mediated gene activation (CRISPRa). The crRNA* and tracrRNA* were expressed under the U6 promotor to ensure the sufficient amount for the trigger‐induced assembling process [46] . HEK‐293T with stably expressed dCas9‐VPR was selected as the model cell line to verify the regulatory effect between two independent genes.…”

A systematic screening assay identifies efficient small guide RNAs for CRISPR activation

“…A second Golden Gate reaction is then used to insert up to four different sgRNA cassettes into lentiviral transfer vectors containing an active Cas9 for gene editing. As this Golden Gate design uses different pol III promoters for each of the sgRNA cassettes, each short RNA will be expressed with different efficiencies (Goguen et al, 2021), potentially causing suboptimal activation, especially for multiplex activation. More recently, Savell and colleagues used Golden Gate to clone up to eight sgRNA cassettes in tandem, all driven by separate human U6 promoters, for multiplex CRISPRa using a dual lentiviral vector system (Savell et al, 2020).…”