A direct comparison of strategies for combinatorial RNA interference
© Lambeth et al; licensee BioMed Central Ltd. 2010
Received: 1 April 2010
Accepted: 11 October 2010
Published: 11 October 2010
Combinatorial 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.
Double-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.
By 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.
Since the first application of DNA-delivered RNA interference (RNAi), the expression of short hairpin RNAs (shRNAs) for targeted gene silencing has become a benchmark technology. Using plasmid and viral vectoring systems, the transcription of double stranded RNA precursors that are processed by the RNAi pathway has lead to potent gene-specific knockdown. Importantly, such strategies can permit the long-term delivery of shRNAs to overcome the limitation of transient suppression by small interfering RNAs (siRNAs). Building upon the early experimental success of expressed shRNAs, the delivery of multiple RNAi effectors, known as combinatorial RNAi (co-RNAi), can offer considerable advantages over the use of single molecule knockdown strategies [reviewed in 1, 2]. Co-RNAi is particularly important for evolving targets that require long-term treatment such as highly mutable RNA viruses like human immunodeficiency virus (HIV) and hepatitis C virus (HCV). Recent studies have shown that the replication of these viruses can be suppressed for periods as long as 75 days by the expression of two of more shRNAs simultaneously [3–5]. In addition, the prospect of increased levels of gene silencing and for multiple-gene targeting is also extremely important for many other transcripts that are not particularly susceptible to spontaneous mutation such as host genes and DNA viruses.
There are currently three main methods to achieve co-RNAi in animal cells; multiple promoter/shRNA cassettes, long hairpin RNAs (lhRNA) and microRNA-embedded shRNAs. The expression of multiple shRNAs from a single construct encoding several separate promoter/shRNA cassettes offers the potential for relatively straightforward vector construction as previously validated RNAi cassettes can be simply assembled as tandem repeats. Recent studies have included the use of shRNA cassettes in combinations of two , three [3, 7, 8], four [4, 9, 10], six [11, 12], and in one study a cloning strategy for the production of up to seven was described but not validated . Results consistently show that such approaches provide an additive effect on single and multiple-gene knockdown on a variety of host and viral gene targets. Although in one study, individual shRNAs were transcribed at much lower levels when expressed from a 4 cassette construct compared to single copy vectors .
The use of lhRNA or extended shRNAs (e-shRNAs) represent a likely progression from single site targeting as long dsRNA are naturally processed as part of the RNAi pathway, and such molecules have been shown not to induce interferon mediated responses [14–16]. A number of recent studies have successfully utilised this approach to target and suppress HIV replication [5, 15, 17–21] and as a result, the parameters that determine efficient processing have been well defined. In particular, the effect of varying siRNA stem length and positioning, spacing between siRNAs stems, and the relative abundance of processed molecules have been tested [19, 20]. Despite these advances, siRNAs have been produced from 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 [5, 19–21].
The insertion of shRNA sequences into naturally occurring microRNA (miRNA) precursor sequences represents a potentially favourable strategy as effector molecules should be processed and exported by the same cellular pathways as endogenous miRNAs. Moreover, it has been found that miRNA mimic shRNAs can abolish competition of siRNAs and shRNAs for transport and incorporation into RISC . The insertion of an effective shRNA into the miR-30 pre-miRNA backbone sequence resulted in enhanced activity  and by embedding shRNAs in repeated miR-30 flanking sequences up to three shRNAs were transcribed from single constructs [24–27]. In addition, the BIC transcript which encodes miR-155, was modified to express multiple shRNAs  and a commercial vector featuring miR-155 flanking sequences and an shRNA cloning site has been widely used for shRNA delivery. The potential for multiple shRNA expression by modifying naturally occurring polycistronic miRNA clusters have also been shown using an endogenous human miRNA cluster  and chicken miRNA cluster .
Previous studies have clearly demonstrated that each co-RNAi strategy can be used to achieve highly effective gene suppression. However, these experiments generally focus on the use of one chosen co-RNAi approach, often involving substantial optimisation of that system. To provide an overall picture of the strengths and weaknesses of each method of co-RNAi, we took a set of active siRNAs and delivered them using validated and proven expression systems without extensive method-specific optimisation. By directly comparing the ability of these shRNAs to initiate knockdown of the same gene targets, this study provides practical information for researchers looking to express multiple validated siRNA sequences using an existing co-RNAi platform.
shRNA and lhRNA plasmids
siRNA and shRNA sequences and their reporter targets used in this study
Target gene (Ref)
MDV gB 
MDV gB 
MDV UL29 
MDV ncRNA (unpublished data)
Renilla luciferase (unpublished data)
IBDV VP2 
miRNA-embedded shRNA plasmids
shRNA, lhRNA and miRNA-embedded RNAi cassettes were cloned into the retrovirus RCASBP-(A)-CN-EGFP (with avian leukosis virus subgroup A envelope, a generous gift from Dr Jon Gilthorpe, Kings College London) using the unique NotI site. Prior to the insertion of the dual-U6/shRNA cassettes into this vector, the chicken U6-4 promoter was amplified from chicken genomic DNA using U6-specific primers as previously described  with an introduced SpeI site in the forward primer (5'-GGACTAGTGAATTGTGGGACGGCGGAAG-3'), and reverse primer (5'-ATCGATGGGGCGCGCCGTTTAAACACTAGTTCGAACCCCAGTGTCTCTCGGACAGTA-3') with introduced BstBI, AscI and ClaI sites for the insertion of shRNA templates. Single U6/shRNA plasmids for the chicken U6-4 promoter were then generated for sh-1b and sh-2 using the same method as described previously . These constructs were then digested with SpeI and ligated into U6-sh1a also digested with SpeI. The entire single and dual-U6/shRNA cassettes and U6/lhRNA cassettes were then excised by digestion with NotI and inserted into RCASBP-(A)-CN-EGFP also digested with NotI. To insert the miRNA-embedded shRNA cassettes, pRFPRNAi-specific primers were used to amplify the U6/miRNA inserts using forward primer (5'-TCGACCTGCAGCCCAAGCTT-3') and reverse primer (5'-ATAAGAATGCGGCCGCGCAGCGGATCCATCGATAAA-3') which contained an introduced NotI site. Amplified fragments were then digested with NotI and inserted into RCASBP-(A)-CN-EGFP that had also been digested with NotI. All U6/RNAi cassettes inserted into RCASBP-(A)-CNEGFP were in the reverse orientation.
Cells and transfections
The DF-1 cell line derived from line 0 chicken embryonic fibroblasts  were maintained in Dulbecco's modified Eagles medium (DMEM) containing 10% foetal calf serum with 10% CO2 at 39°C and used for reporter assays and for retrovirus growth. Plasmid DNA and siRNA transfections were carried out in 96-well plates using 125 ng of each plasmid DNA or 50 nM siRNAs using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. For retrovirus growth, 2 μg of RCAS-U6/shRNA plasmid DNA was transfected into DF-1 in 6-well plates and passaged 6 days post-transfection into T75 flasks for RNA isolation or 96-well plates for reporter plasmid transfection.
The reporter vector psiCHECK™-2 carrying the sequences of the various shRNA target transcripts were assayed for luciferase expression using the Dual Glo Luciferase Assay System (Promega) following the manufacturer's instructions. The relative expression of target specific Renilla luciferase was determined by taking the normalised levels compared to background Firefly luciferase for each sample transfected in four replicates ± standard error and is representative of at least two independent experiments.
Total RNA was extracted from cultured DF-1 cells infected with retroviral vectors using TRIzol reagent (Invitrogen) according to standard methods described by the manufacturer. Samples of 30 μg total RNA were resolved using a 15% polyacrylamide-1 × Tris-borate-EDTA-8 M urea gel and blotted to a GeneScreen Plus membrane (Perkin-Elmer). DNA oligonucleotides with sequences complementary to the shRNAs were end labelled with [γ-32P] ATP (Amersham) and T4 polynucleotide kinase (New England Biolabs) to generate high-specific-activity probes. Hybridization, washing, and autoradiography were carried out as previously described .
Experimental data was analysed for statistical significance using two-tailed unpaired T-tests, where P values of less than 0.05 were considered significant. Statistical analysis was carried out using GraphPad Prism version 5.0b.
Efficacy of single and double-gene targeting U6 expressed shRNAs
The simultaneous expression of several RNAi effectors can be achieved using various approaches, including multiple promoter/shRNA cassettes, long hairpin RNAs (lhRNA) and microRNA-embedded shRNAs (Figure 1). For the direct comparison of expressed shRNA from each co-RNAi platform, it was important to first test that the set of already validated siRNA sequences could be expressed as standard 19-nt shRNAs to achieve similar levels of gene knockdown. The sequences of all siRNAs and their target genes are detailed in Table 1, and all shRNAs and their target reporter plasmids are shown in Figure 2A. Co-transfection of reporter plasmids with either siRNAs or their equivalent shRNAs showed that for each of the four different molecules tested the efficacy of the expressed shRNA was very similar to its equivalent siRNA (Figure 2B), indicating that they were appropriate for use in the comparison of co-RNAi methods.
Efficacy of single and double-gene targeting U6 expressed long hairpin RNAs
Efficacy of single and double-gene targeting U6-expressed miRNA-embedded shRNAs
Comparison of co-RNAi methods by retroviral delivery
To determine which of the three co-RNAi methods represents the most effective and robust strategy for gene suppression, we directly compared the most efficient single and double-gene knockdown vectors for their ability to suppress reporter gene activity. To avoid the inconsistencies associated with co-transfection of RNAi and reporter plasmids, and to provide a background that is more applicable to experimental gene knockdown studies, we inserted the most effective of each dual RNAi cassette into the retroviral vector RCASBP(A)-CN-EGFPm5. However, Considering that the recombination and deletion of repeated U6 sequences in viral vectors has been seen before [4, 41], prior to insertion of the dual-U6 cassettes into RCAS, we swapped the second U6 promoter of these constructs with the chicken U6-4 promoter. It was found that sh-1b and sh2 expressed from this promoter achieved a similar level suppression to those transcribed by chU6-3 (data not shown), a finding that is consistent with others [33, 42].
Analysis of the limitations of co-RNAi
The limitations of lhRNA-mediated RNAi are evident both in previous studies and by the data presented in the current study. It has been shown that the siRNA activity diminishes with distance from the lhRNA base [19–21], and we showed that the efficacy of the three siRNA tested varied depending on the sequence and location within the lhRNA (Figure 4A and 4C). Taken together, it appears that although the use of lhRNA can result in highly effective gene knockdown, this might require detailed optimisation for a given set of sequences, rather than simply linking effective siRNAs sequences together.
In the current study, it was shown that cassette location and orientation in the dual-U6/shRNA vectors did not markedly affect shRNA activity. However, a previous report found that the abundance of individual shRNAs expressed from similar constructs decreased with when four shRNA cassettes were present , although the effect of this on gene suppression was not tested. To further explore these observations, we generated a series of vectors featuring increasing numbers of U6/shRNA cassettes and tested their ability to induce reporter gene knockdown (Figure 7B). A total of five U6/shRNA cassettes targeting five different reporter sequences were developed, allowing for the analysis of individual shRNA efficacy from vectors carrying 1, 2, 3, 4 or 5 separate U6/shRNA cassettes. Co-transfection of the multiple U6/shRNA plasmids along with appropriate reporter vectors showed that all of these were able to suppress gene expression at a rate similar to that of the positive controls (single U6/shRNA) regardless of how many cassettes were present. In particular, the shRNAx5 vector induced comparable levels of gene suppression to each of the single U6/shRNA positive controls, except for sh-4 however, which showed a 20% reduction in activity. A reduction in activity was also evident for the shRNAx3 vector when targeting the psi-CHK-3 reporter, and the shRNAx5 when targeting the psi-CHK-4 reporter, as both were slightly less effective compared to their respective positive control shRNAs. Importantly however, reporters 1, 2 and 5 showed equally efficient knockdown regardless of the number of shRNA cassettes present. Overall, these data suggest that vectors encoding up to five U6 promoters can maintain very high levels of individual gene silencing activities.
Co-RNAi has been used to achieve potent gene silencing of single and multiple-gene targets by means of a number of different techniques. Although all of these methods can result in efficient gene suppression, such studies often involve the detailed optimisation of the chosen system and experimental findings are not reported in the context of the other delivery options. Since the initial use of pol III expressed shRNAs in mammalian cells [43, 44] this strategy has become a standard technique for single-target vector delivered RNAi and has been the subject of extensive optimisation. More recently, the analysis of key factors that determine lhRNA and miRNA-embedded shRNA efficacy, has seen both of these strategies achieve enhanced gene knockdown. For lhRNAs this has included siRNA length and spacing between siRNAs stems [19, 20], and for miRNA-embedded shRNAs the use various flanking sequences and lengths [26, 28], and the placement  of siRNAs within miRNA sequences contexts has been tested.
Following the identification of effective siRNA molecules for target genes of interest, the selection of a delivery strategy for co-RNAi presents several options, each of which appear to offer reliable gene knockdown. To determine which of these methods is the most effective, predictable and robust, we took a set of active siRNAs and directly compared their ability to suppress gene expression when delivered by each co-RNAi platform. Since the goal of this study was also to determine the ease at which each technique could be applied to any given set of siRNAs, we used standard 19-nt siRNAs rather than molecules that have been specifically optimised and selected for each individual expression system. This study therefore analyses the adaptability of standard siRNA sequences that would be generated by publicly available algorithms or as custom designed siRNAs to be used in each of the delivery platforms described. Prior to testing the siRNAs in the co-RNAi vectors, the expression of these sequences as regular 19-nt shRNAs resulted in equivalent gene suppression (Figure 2B), illustrating that these particular siRNAs can be effectively expressed as shRNAs without optimisation. Initially, we analysed dual shRNA expression approaches as previous experiments suggest that although using three and four shRNA expression constructs can be highly effective, these only slightly improve on the levels of gene silencing seen by the dual systems [5, 29]. In addition, the levels of shRNA generated from multiple promoter constructs diminished when numerous promoters were used , and the second, third and fourth siRNA in lhRNA constructs become increasingly less effective the further they are away from the stem [5, 19, 20]. Therefore, to keep the number of variables at a minimum, this study initially involved the comparison of multiple promoter/shRNA, lhRNAs and miRNA-embedded shRNA vectors expressing only two siRNAs targeting either single or double gene combinations.
Our data showed that both dual-U6/shRNA and miRNA-embedded expression plasmids increased gene suppression of single-gene targets compared to equivalent to single shRNAs. In contrast, the single-gene targeting lhRNAs only suppressed gene expression to levels equivalent to the first siRNA, which is not surprising considering the reported gradient-effect in which they are produced. For the double-gene targeting plasmids, both lhRNA and miRNA-embedded shRNAs were more variable and showed slightly decreased gene knockdown compared to the dual-U6/shRNA plasmids, which performed equally well as single shRNAs. Using retroviral-delivered co-RNAi, each expression platform was directly compared showing that although the single-gene targeting miRNA-embedded vector was reasonably effective, it was only the dual-U6/shRNA vectors that achieved a similar level of both single and double reporter gene knockdown to the control shRNAs. However, considering that these vectors were not able to provide enhanced gene knockdown (as seen in the co-transfections in Figure 3A), overall the dual-U6/shRNA constructs did not provide a substantial increase in activity compared to single U6/shRNAs. The lack of activity of the lhRNA retroviruses was explained by the appearance of unprocessed RNA precursors by Northern blotting, and the presence of faint bands for the miRNA-embedded shRNAs was consistent with their low activity in the reporter assays. These data showed that for this set of siRNAs, despite using slightly optimised configurations for both lhRNA and miRNA-embedded shRNA, that the dual-U6/shRNAs provided the most effective and robust gene silencing.
By comparing the efficacy of different siRNA molecules expressed as lhRNAs or miRNAs, it appeared that both the location and individual properties of each siRNA could affect silencing activity. Perhaps qualities such as sequence and local structure of different molecules can directly impact upon efficient processing and production of siRNAs, possibly through changes in their thermodynamic properties. For example, when expressed as an lhRNA, sh-1a was most effective in the first position, whereas sh-2 was more effectual in the second position. Furthermore, when expressed as miRNA-embedded shRNAs, sh-1a was most efficient in position 2, whereas sh-2 which was most effective in position 1. To further explore this, we examined the individual efficacy of three different shRNAs expressed from each of the locations as embedded-miRNAs and found enormous variation ranging from increased to completely abolished activity. This suggests that the individual sequence of each shRNA can have a marked impact on the processing and subsequent activity of shRNAs expresses from each loci of this vector. Unlike lhRNAs and miRNA-embedded shRNAs, U6-transcribed regular 19-nt shRNAs do not appear to be affected by their surrounding sequences. However, since reduced levels of individual shRNAs is caused by increasing numbers of RNAi cassettes , we tested if this reduction translated to a functional effect on levels of gene knockdown. Overall, the expression of up to 5 separate shRNAs from a single construct resulted in a small decrease in activity for only one of the five targets, suggesting that although each shRNA might be less abundant, there was still a sufficient amount to reduce gene expression to similar levels. One concern over the use of multiple promoters for co-RNAi, and indeed strong promoters such as U6 in general, is the saturation of the RNAi machinery resulting in impaired processing of endogenous miRNAs [22, 45, 46]. These data would suggest however, that since pol III transcribes individual shRNAs from multiple promoter constructs at lower levels than single promoter cassettes, the overall levels of shRNAs in these cells may be similar to those containing single cassettes and may therefore not further add to this concern.
The parameters that determine shRNA efficacy are now very well defined; the same cannot be said however, for constructs that express multiple RNAi effectors. By directly comparing the same set of siRNAs expressed using three separate methods of co-RNAi we have identified some of the strengths and pitfalls associated with each technique. Overall, the use of multiple U6/shRNA cassettes offered the most reliable and predictable gene knockdown of both single and multiple-gene targets, an effect that was not inhibited by having up to five separate shRNA cassettes. It remains to be seen however, the impact of having multiple pol III promoters on cellular RNAi machinery, endogenous miRNA processing and the expression of native pol III transcripts. In such a case, the use of RNAi effectors that resemble naturally occurring molecules such as miRNAs and long dsRNAs, may best avoid unwanted cellular effects, especially considering that miRNA mimic shRNAs can abolish competition of siRNAs and shRNAs for transport and incorporation into RISC . In any case, this study for the first time directly compares three methods for the delivery of multiple shRNAs and provides valuable insights for the design and application of reliable combinatorial RNAi.
This work was supported by the Biotechnology & Biological Sciences Research Council (BBSRC), UK.
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