Harnessing Intronic microRNA Structures to Improve Tolerance and Expression of shRNAs in Animal Cells

Exogenous RNA polymerase III (pol III) promoters are commonly used to express short hairpin RNA (shRNA). Previous studies have indicated that expression of shRNAs using standard pol III promoters can cause toxicity in vivo due to saturation of the native miRNA pathway. A potential way of mitigating shRNA-associated toxicity is by utilising native miRNA processing enzymes to attain tolerable shRNA expression levels. Here, we examined parallel processing of exogenous shRNAs by harnessing the natural miRNA processing enzymes and positioning a shRNA adjacent to microRNA107 (miR107), located in the intron 5 of the Pantothenate Kinase 1 (PANK1) gene. We developed a vector encoding the PANK1 intron containing miR107 and examined the expression of a single shRNA or multiple shRNAs. Using qRT-PCR analysis and luciferase assay-based knockdown assay, we confirmed that miR30-structured shRNAs have resulted in the highest expression and subsequent transcript knockdown. Next, we injected Hamburger and Hamilton stage 14-15 chicken embryos with a vector encoding multiple shRNAs and confirmed that the parallel processing was not toxic. Taken together, this data provides a novel strategy to harness the native miRNA processing pathways for shRNA expression. This enables new opportunities for RNAi based applications in animal species such as chickens.

Automated summary

Exogenous RNA polymerase III (pol III) promoters are commonly used to express short hairpin RNA (shRNA). However, their expression can cause toxicity due to saturation of the native miRNA pathway. In this study, the researchers developed a novel strategy to mitigate shRNA-associated toxicity by harnessing native miRNA processing enzymes. They found that using miR30-structured shRNAs resulted in the highest expression and subsequent knockdown of target transcripts. Injection of a vector encoding multiple shRNAs into chicken embryos confirmed that this parallel processing was not toxic. This study provides new opportunities for RNAi-based applications in animal species.