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Orthogonal design in software and RNAi screening

Orthogonal design in software and RNAi screening

The software engineering classic The Pragmatic Progammer popularised the benefits of orthogonality in software design.  They introduce the concept by describing a decidedly non-orthogonal system:

You’re on a helicopter tour of the Grand Canyon when the pilot, who made the obvious mistake of eating fish for lunch , suddenly groans and faints. Fortunately, he left you hovering 100 feet above the ground. You rationalize that the collective pitch lever [2] controls overall lift, so lowering it slightly will start a gentle descent to the ground. However, when you try it, you discover that life isn’t that simple. The helicopter’s nose drops , and you start to spiral down to the left. Suddenly you discover that you’re flying a system where every control input has secondary effects. Lower the left-hand lever and you need to add compensating backward movement to the right-hand stick and push the right pedal. But then each of these changes affects all of the other controls again. Suddenly you’re juggling an unbelievably complex system, where every change impacts all the other inputs. Your workload is phenomenal: your hands and feet are constantly moving, trying to balance all the interacting forces.

[2] Helicopters have four basic controls. The cyclic is the stick you hold in your right hand. Move it, and the helicopter moves in the corresponding direction. Your left hand holds the collective pitch lever. Pull up on this and you increase the pitch on all the blades, generating lift. At the end of the pitch lever is the throttle . Finally you have two foot pedals, which vary the amount of tail rotor thrust and so help turn the helicopter.

As the authors explain:

The basic idea of orthogonality is that things that are not related conceptually should not be related in the system. Parts of the architecture that really have nothing to do with the other, such as the database and the UI [user interface], should not need to be changed together. A change to one should not cause a change to the other.

This applies to many types of design, not just for computer systems.  The plumber should not have to depend on the electrician to fix a broken pipe.

The principle has also been used in RNAi screening, notably by Perreira et al. who introduce the MORR (Multiple Orthologous RNAi Reagent) method to increase confidence in screening hits.  Comparing the results of siRNAs from different manufacturers  is important, but because they operate by the same mechanism (including the off-target effect), they are not really orthologous.  More orthologous would be the comparison between RNAi and CRISPR experiments, which sometimes show discrepancies that point to interesting biology.

To confirm RNAi screening hits, ‘partial orthogonality’ may be preferable.  If screening hits are due to either on-target or off-target effects, confirmation with RNAi reagents that only have one or the other would be better than using CRISPR, where it is difficult to interpret the reason for discrepancies (e.g. is there no phenotype  because of genetic compensation?).

One could use C911s to create a version of the siRNA that, in theory, maintains off-target effects but eliminates on-target effects.  We have observed, however, that C911s often give substantial knockdown of the original target gene (in some ways, C911s are like very good microRNAs).  To be sure that a positive effect with C911s is not due to partial knockdown, one would also need to test that via qPCR.  C911s can create a lot of work.

Far better would be to confirm screening results with siPOOLs, which provide robust knockdown and minimal off-target effects.

One place RNAi practitioners would hope not to find orthogonality is the relationship between on-target knockdown and phenotypic strength.

Since the early days of RNAi, positive correlation between knockdown and phenotypic strength has been suggested as a means to confirms screening results.  Reagents with a better knockdown should give a stronger phenotype.

To test this, we obtained qPCR data for over 2000 siRNAs (Neumann et al.) and checked the performance of those siRNAs against the designated hit genes from an endocytosis screen (Collinet et al.).

If the siRNAs work as expected, those siRNAs with better knockdown should give stronger phenotypes than those with weaker knockdown.

There were 100 genes from the Collinet hits for which there were 3 siRNAs with qPCR data.

For those 100 siRNAs triplets, we compared the phenotypic ranks with the knockdown ranks.  (We were agnostic about the direction of phenotypic strength, and checked whether knockdown and phenotype were consistent when phenotype scores were ranked in either ascending or descending order).  For example, if siRNAs A, B, and C have phenotypic scores of 100, 90, 70 and knockdown of 15%, 20%, 30% remaining mRNA, we would say that phenotypic strength is consistent with knockdown (and because we were agnostic about phenotypic direction, we would also say it was consistent if siRNAs A, B, and C had scores of 70, 90, 100).

The observed number of cases where knockdown rank was consistent with phenotypic rank was then compared to an empirical null distribution, obtained by first randomising the knockdown data for the siRNA triplets before comparison to phenotypic strength.  This randomisation was performed 300 times.  This provides an estimate of what level of agreement between knockdown and phenotype would be expected by chance.  The standard deviation (SD) from this null distribution was then used to convert the difference between observed and expected counts into SD units.

The Collinet dataset provides data for 40 different features.  The above procedure was carried out for each of the 40 features.

To take one feature (Number vesicles EGF) as an example, we observed 34 cases where knockdown was consistent with phenotypic strength.  By chance, we would expect 33.4 (with a standard deviation of 4.9).  The difference in SD units is (34-33.4)/4.9 = 0.1.

As can be seen in the following box plot, the number of SD units between observed and expected counts of knockdown/phenotype agreement for the 40 features is centered near zero (median is 0.1 SD units):

This suggests that there is very little, if any, enrichment in cases where siRNA knockdown strength is correlated with phenotypic strength.  The orthogonality between knockdown and phenotype, given the poor correlation between siRNAs with the same on-target gene, is unfortunately not unexpected.

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siRNA vs shRNA – applications and off-targeting

siRNA vs shRNA – applications and off-targeting

Short interfering RNA (siRNA) and short hairpin RNA (shRNA) are both used in RNAi-mediated gene silencing. In this blogpost, we explore the differences in applications of siRNA and shRNA and compare their capacity for off-targeting.

For a summary of their properties, please refer to Table 1 at the end of  the post.

In what situations should we use siRNA or shRNA?

In terms of application, siRNAs are commonly applied for rapid and transient knockdown of gene expression.

It is performed in cell lines amenable to transfection by liposomes/electroporation and effects typically last from 3-7 days though retransfection can be performed to extend the effect.

The amount of siRNA introduced can be highly controlled and efficiency of gene knockdown is dependent on the levels of siRNA in the cell which is influenced by transfection efficiency and siRNA stability. Knockdown is also influenced by characteristics of the gene. A gene that is highly transcribed for example, may experience less siRNA-mediated downregulation compared to a gene where lesser copies of RNA are produced over time. In addition, a gene which expresses a protein with a very long half-life, may require extended periods of siRNA application to see a knockdown effect.

Due to the transient effect of siRNAs, shRNAs were developed to be used for prolonged knockdown of genes.

As they are introduced by viral vectors, cells that are more difficult to transfect are better targeted with shRNA. Furthermore, promoter-driven expression allows for inducible expression of the shRNA. Depending on the viral vector used – refer to Labome’s post that covers siRNA/shRNA delivery in greater detail – the shRNA may be integrated into the host genome, allowing it to be propagated into daughter cells. This maintains a consistent gene knockdown over several generations. However, knockdown efficiency can decline over time. This is mainly due to varying levels of uptake of the shRNA among cells, with a cell population having lower shRNA expression being over-represented with time.

 

What about RNAi screening?

siRNAs and shRNAs are both used in RNAi screening to identify genes of interest in a studied phenotype. These are performed with siRNA/shRNA libraries that target a large variety of genes. There are two RNAi screening formats commonly used – arrayed and pooled.

siRNAs and shRNAs can both be used in an arrayed screening format. This means that the siRNA(s)/shRNA(s) against each gene is tested in distinct cell populations. Arrayed screens have the advantage of being compatible with various phenotypic readouts and do not suffer from possible reagent cross-talk or challenges associated with deconvoluting data. However, they are more energy and resource-intensive to perform. (See Fig. 2)

The pooled screening format in contrast, applies only with shRNAs. Here, all shRNAs (e.g. a whole-genome shRNA library) are introduced to a single cell population. As low titers of viral vectors are used, each cell in the population is expected to take up one shRNA vector.

With pooled screening, only readouts linked to cell number can be assessed. These include measurements for cell viability or altered expression of a cell surface marker assessed by fluorescence activated-cell sorting. shRNAs targeting genes which impact these readouts are expected to skew the cell population, such that only cells affected by the relevant shRNAs can be identified. This is either through negative selection, where lost cell populations are noted, or positive selection, where cells with certain shRNAs become over-represented.

The resulting cell population is then assessed by PCR, microarray hybridization or next generation sequencing to measure which shRNAs are highly or lowly-represented. The shRNAs are identified usually by means of a DNA barcode present in the vector sequence. Of note, pooled screens take up less resources to perform but require longer assay times to allow for significant changes in the overall cell population to occur.

Fig. 2 Simplified workflow for arrayed and pooled RNAi screening formats

 

Off-target effects with shRNAs?

The use of siRNAs are known to produce several off-target effects but what about shRNAs? Given they are processed the same way as siRNAs, shRNAs are also subject to microRNA-like off-target effects. In addition, because they are expressed from DNA and rely on endogenous machinery to be processed into siRNA, several variations may be introduced not found with introducing siRNA directly. Some potential sources of off-target effects for shRNAs include:

1. Promoter-driven expression. shRNAs are typically controlled with a U6 promoter which drives high levels of transcription via RNA polymerase III. The high shRNA expression levels may saturate endogenous RNAi machinery, contributing to off-target effects. To counter this, shRNAs can be expressed in a context mimicking miRNAs, utilizing RNA polymerase II for transcription instead. This has been found by several groups to reduce the incidence of off-target effects (Grimm et al., 2006, Kampman et al., 2015)

2. Dicer-mediated hairpin processing. shRNAs undergo Dicer-mediated cleavage in the cytosol to remove its hairpin loop. Gu et al., 2012 reported that Dicer cleaves with sufficient heterogeneity to generate multiple sequences. This factor was reported to generate the higher noise levels unique to shRNA screens (Bhinder and Djaballah, 2013). As specificity of Dicer cleavage is influenced by neighbouring loop and bulge structures, care should be taken in shRNA design.

3. Multiple shRNA uptake. During viral transduction, the viral titer is minimized to increase the probability that cells take up a single shRNA vector. However, this does not guarantee that multiple shRNA uptake will not occur. In this event, a combinatorial gene knockdown ensues resulting in a mixed phenotype that may generate false hits.

4. Differences in genomic integration between shRNAs. Varying efficiencies in transfection and genome integration between shRNAs may skew results to over-represent certain shRNAs over others, especially in pooled screens. Furthermore, integration into the host genome may disrupt the function of certain genes, producing more off-targets.

Studies comparing results from siRNA and shRNA screens have found extremely poor overlap, both between and within the reagent-specific screens. Bhinder and Djaballah’s (2013) analysis of results from 30 published RNAi screens (16 siRNA, 14 shRNA) searching for genes that impact cell viability saw no common genes identified across the board. Furthermore, different genes were identified depending on whether the screen used siRNA or shRNA. PLK1 for example, was a prominent hit for siRNA screens but was only marginally represented in shRNA screens. In contrast, KRAS was a top hit among shRNA screens.

Fig. 3 Reagent format of RNAi screens analysed in Bhinder and Djaballah, 2013 Screens were performed either with genome-wide (GW) or focused (FD) siRNA/shRNA libraries. For siRNA screens, Pooled refers to pools of 3 siRNAs applied together compared to Singles where a single siRNA duplex was applied. For shRNA screens, Pooled refers to a pooled format screen (Fig. 2) where ~50, 000 shRNAs were applied to a single cell population. Arrayed refers to arrayed format screen where shRNAs were applied individually (Fig. 2).

Fig. 4 Overlap of hits among genome-wide (left) and focused (right) siRNA screens (Bhinder and Djaballah, 2013) Only 4 common hits detected across the 2 lethal gene lists from genome-wide siRNA screens. In focused siRNA screens, a greater overlap was detected but still limited across the 22 lethal gene lists. PLK1 detected in 9 out of 22 gene lists.

Fig. 5 Overlap of hits among genome-wide (left) and focused (right) shRNA screens (Bhinder and Djaballah, 2013) KRAS was a top hit in shRNA GW screens, appearing in 5 out of 9 lists. In focused shRNA screens, KRAS was present in 15 out of 31 lists. 

Worryingly, an enrichment of gene candidates exclusive to pooled shRNA screens was observed as opposed to arrayed shRNA or siRNA screens. Most of the overlap seen in gene lists (80% global overlaps, 60% after stringent filtering) were specific to pooled shRNA screens. Exclusion of data from pooled shRNA screens would have reduced overlap to a mere 27%. This indicates gene targets obtained from shRNA pooled screens is specific to the technique as opposed to specific gene downregulation.

Furthermore, a greater number of hits were obtained from shRNA screens – 6664 candidates from 40 shRNA gene lists – as opposed to 1525 candidates from 24 siRNA gene lists. This indicates a generally noisier dataset associated with shRNA screens.

Bhinder and Djaballah later performed a head-to-head comparison of an arrayed siRNA and shRNA screen and reported similarly dismal results. Despite using a gain-of-function assay, which tends to yield clearer results, only a 29 hit overlap was seen between siRNA and shRNA libraries which shared 15,068 common genes. Based on a known set of positive controls, siRNAs identified 8 known regulators as opposed to shRNA which only identified 3. Furthermore, predicted siRNA sequences obtained after Dicer-processing of shRNA which corresponded to exactly the same siRNA sequence from the siRNA library yielded different phenotypes. The authors highlight that differential intracellular processing of the shRNA contributes significantly to the discrepancies observed.

It is evident that shRNAs are at risk to greater number of off-target effects than siRNAs. Much care should be taken towards the interpretation of pooled shRNA screens in particular. Secondary validation of gene hits plays an increasingly important role. It is recommended to validate gene hits with siPOOLs (high-complexity, defined siRNA pools) which have a lower off-target profile than single siRNAs or low complexity siRNA pools of 3-4. siPOOL-resistant rescue constructs enable further affirmation that the loss-of-function phenotype is attributed to the target gene. Alternative tools such as compounds, antibodies or gene knockout technologies are also highly recommended.

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Table. 1 Comparison of properties between siRNA and shRNA

siRNA shRNA
Structure 20-25 nucleotide long double-stranded RNA (dsRNA) with 2 nucleotide overhangs at the 3’ end

~57-58 nucleotide long RNA sequence with a dsRNA region linked by non-pairing nucleotides to form a stem-loop structure

Delivery RNA itself with liposome/electroporation-mediated delivery into cells Usually delivered to cells via viral vectors. DNA may be incorporated into host genome depending on viral vector used.
Processing In the cytosol, guide or antisense strand* (shown in blue in Fig. 1) is incorporated into RNA induced silencing complex (RISC). RISC is guided towards RNA transcripts with the complementary sequence to mediate cleavage and subsequent degradation of the transcript.

 

*Note that the sense strand may also load into RISC and mediate off-targeting but incidence of this is reduced by designing siRNA with  appropriate thermodynamic properties (refer to previous blogpost on siRNA design)

In the nucleus, shRNA is transcribed from DNA by either RNA polymerase I or III, depending on the promoter.

Drosha, a member of the ribonuclease III family, processes the RNA transcript of its long flanking single-stranded RNA sequences and the resultant shRNA is exported out of the nucleus by Exportin-5.

 

In the cytosol, the enzyme Dicer cuts off the hairpin loop of the shRNA and releases the functional active siRNA which follows the same downstream processing as siRNAs.

 

Length of expression Varies from 3-7 days. Affected by degradation of siRNA within cell and dilution of effect upon cell division. Expression can be reinstated by re-transfecting the siRNA. If the DNA is stably integrated in the host genome, knock-down is theoretically permanent.
Control of knockdown Easily controlled by varying amount of siRNA introduced. Magnitude of knockdown harder to control as determined by promoter-driven efficiency and shRNA vector uptake. Expression however can be made inducible with Tet-on/off systems.

 

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