A direct comparison of strategies for combinatorial RNA interference

Abstract

BackgroundCombinatorial RNA interference (co-RNAi) is a valuable tool for highly effective gene suppression of single and multiple-genes targets, and can be used to prevent the escape of mutation-prone transcripts. There are currently three main approaches used to achieve co-RNAi in animal cells; multiple promoter/shRNA cassettes, long hairpin RNAs (lhRNA) and miRNA-embedded shRNAs, however, the relative effectiveness of each is not known. The current study directly compares the ability of each co-RNAi method to deliver pre-validated siRNA molecules to the same gene targets.ResultsDouble-shRNA expression vectors were generated for each co-RNAi platform and their ability to suppress both single and double-gene reporter targets were compared. The most reliable and effective gene silencing was achieved from the multiple promoter/shRNA approach, as this method induced additive suppression of single-gene targets and equally effective knockdown of double-gene targets. Although both lhRNA and microRNA-embedded strategies provided efficient gene knockdown, suppression levels were inconsistent and activity varied greatly for different siRNAs tested. Furthermore, it appeared that not only the position of siRNAs within these multi-shRNA constructs impacted upon silencing activity, but also local properties of each individual molecule. In addition, it was also found that the insertion of up to five promoter/shRNA cassettes into a single construct did not negatively affect the efficacy of each individual shRNA.ConclusionsBy directly comparing the ability of shRNAs delivered from different co-RNA platforms to initiate knockdown of the same gene targets, we found that multiple U6/shRNA cassettes offered the most reliable and predictable suppression of both single and multiple-gene targets. These results highlight some important strengths and pitfalls of the currently used methods for multiple shRNA delivery, and provide valuable insights for the design and application of reliable co-RNAi.

Short Hairpin RNA-Mediated Gene Silencing

“…Despite these advances, siRNAs have been produced from both e-shRNA and lhRNA precursors in a gradient with the most abundant and active being at the end distal from the loop, resulting in reduced silencing for the second, third, and fourth siRNAs (128)(129)(130)(131) . It was also found that compared to other methods of co-RNAi, the same shRNA sequences were much less predictable when expressed as e-shRNAs and generally exhibited reduced silencing activity ( 141 ) . Nonetheless, these examples clearly show that these approaches can be very successfully used to initiate potent gene knockdown, particularly in the case of HIV.…”

“…when expressed from a 4-cassette construct compared to single copy vectors ( 5 ) , but in a separate study, vectors encoding up to fi ve separate promoter/shRNA cassettes offered similar levels of gene knockdown compared to single shRNAs ( 141 ) . One potential hazard of this strategy was identi fi ed by the unwanted recombination and loss of identical promoter sequences cloned into lentiviral vectors, an event that was then avoided by using different promoter sequences ( 5 ) .…”

“…Two recent studies attempted to shed light on this inconsistency by comparing various co-RNAi methods directly to one another. Both essentially concluded that the use of multiple promoter/shRNA cassettes encoded in a single vector offered the most useful approach for immediate application in gene therapy ( 20,141,151,152 ) . This was attributed to the predictable and effective nature of these constructs, the simplicity of vector construction, and the ability to use existing shRNA sequences without further optimizations.…”

Oligonucleotide-Based Theranostic Nanoparticles in Cancer Therapy

“…Cassettes that express multiple shRNA sequences allow for a combinatorial RNA interference approach, resulting in greater gene suppression than with single-shRNA cassettes [86][87][88][89]. The efficacy of shRNAs can be improved by incorporating mutations and increasing stem length [86,[90][91][92].…”

Generation of Genetic Constructs that Simultaneously Express Several shRNAs

“…This so-called combinatorial RNA interference (co-RNAi) is especially important for treating highly mutable viruses such as HIV-1 because they tend to escape from RNAi (2). There are three main strategies for using co-RNAi in antiviral gene therapy: (1) multiple promoter/shRNA cassettes; (2) long hairpin RNAs; and (3) microRNA-embedded shRNAs (35). In this paper we describe a protocol for generation of shRNA cassettes that express several shRNAs under a single promoter in human cultured cells.…”