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Month: July 2017

CRISPR/Cas9 Screening – The “Copy-Number Effect”

CRISPR/Cas9 Screening – The “Copy-Number Effect”

Several CRISPR/Cas9 screens identifying essential genes in cancer cell lines have been performed to date (Shalem et al., 2014, Hart et al., 2015, Kiessling et al., 2016). These typically take the form of pooled screens where sgRNA libraries targeting all genes or subsets of genes are introduced in parallel into Cas9-expressing cells, at a single sgRNA per cell. The sgRNAs exert a negative or positive selection pressure on cells based on their impact on cell viability and proliferation. The most depleted or enriched sgRNA sequences are determined by next-generation sequencing, revealing relevant gene ‘hits’. Very similar to how pooled shRNA screens are performed.

From these screens, several groups have observed a worrying phenomenon: CRISPR gRNAs targeting genomic regions of high copy number amplification showed a striking reduction in cell proliferation/survival. Dr William Hahn’s group at the Dana Farber Institute was one of the first to characterize this in a publication last year involving a CRISPR/Cas9 screen on 33 cancer cell lines looking for essential genes. In total, 123411 unique sgRNAs were used targeting 19050 genes (6 sgRNAs/gene), 1864 miRNAs and 1000 non-targeting negative control sgRNAs.

What they discovered is a little worrying to say the least.

The figure shows two genomic regions in two different cell lines (SU86.86 and HT29). At genomic coordinates highlighted by the red box, 3 tracks are shown. Top, copy number from the Cancer Cell Line Encyclopaedia (CCLE) SNP arrays, red indicating above average ploidy and blue showing below; middle, CRISPR/Cas9 guide scores with purple trend line indicating the mean CRISPR guide score for each CN segment defined from the above track; bottom, RNAi gene-dependency scores. AKT2 and MYC, known driver oncogenes at these loci, respectively, are highlighted in orange. For RNAi data, shRNAs targeting AKT2 used in Project Achilles were not effective in suppressing AKT2 (hence the negative result).

 

Key findings:

  • A striking enrichment of negative CRISPR guide scores (i.e. sgRNAs that reduced cell proliferation/survival) for genes that reside in genomic regions of high copy-number amplification.

 

  • Genes identified in CRISPR that reduced survival, did not have the same effect when disrupted by RNAi in the same cell lines (this RNAi screen was done by the same group but published 2 years before).

 

  • This enrichment was seen also for unexpressed genes, i.e. genes not transcribed. Meaning the reduced survival was not due to loss-of-function of the targeted gene.

 

  • Even for regions with low absolute copy numbers, a significant reduction in survival was observed compared to non-targeting control sgRNAs. Furthermore, the effect was dose-dependent with greater copy number amplifications producing larger negative CRISPR guide scores.

Notably, the correlation between copy number and genes that were scored high on essentiality was also observed when looking at data from other studies (Hart et al., 2015). The “copy number effect” would therefore produce a high number of false positives in CRISPR screens for essential genes in cancer cell lines. The graph above shows just how big an effect this is. Comparing genes identified as essential in a CRISPR screen vs RNAi screen, increasingly essential CRISPR-identified genes were more likely to reside on copy number amplifications (defined as having average sample ploidy > 2). This effect was notably absent for RNAi-derived essential genes.

Aside from false positives, the increased noise due to “copy number effects” also increases false negatives. MET, a gene identified by shRNA screens, for example, failed to be picked out by CRISPR screens as it is located on a chromosome 7 amplicon (7q31) in MKN45 cells (gastric cancer cell line) where all other gRNAs within that amplicon also scored as essential.

The authors go on to explore mechanisms behind the “copy number effect”. They found it was attributed to a DNA damage response stimulated by excessive cutting by Cas9. This response appeared p53-dependent and induced cell cycle arrest at the G2 phase, explaining the anti-proliferative effect. A similar response was seen for promiscuous sgRNAs that cut at multiple sites, with effects being more pronounced when cuts were spread over several chromosomes as opposed to a single chromosome.

How to manage this?

So far, most simply avoid analysing hits where sgRNAs lie at amplified regions or target multiple sites (Wang et al., 2017). However, these regions of copy number amplifications have been implicated in cancer and may contain relevant hits. Several computational methods have therefore recently been developed to correct for “the copy number effect”. Hahn’s group developed a computational algorithm called CERES based on data obtained from CRISPR sgRNA screens in 342 cancer cell lines representing 27 cell lineages.

Novartis also developed a Local Drop Out (LDO) algorithm that corrects obtained data based on examining gRNAs scores at direct genomic neighbours. When multiple neighbouring genes show similar drop out scores, effects are assumed to be due to “copy number effects”. This method has the advantage of not requiring prior knowledge of copy number, however it does require a sufficient density of gRNAs to accurately capture “copy number effects”.  They also had an alternative method, Generalized Additive Model (GAM) where copy number was taken into account.

 

How the CERES Model Works

The Results – copy number dependency is reduced while preserving essentiality of cancer-specific genes such as KRAS

 

A step towards the right direction but the penetrance of this effect still raises some concerns:

  • Although false positives are reduced with these computational methods, it is difficult to recapture false negatives. This is dependent on the gRNA having a stronger phenotype compared to neighbouring gRNAs on the amplicon which is not always the case. The LDO method for example still failed to recapture MET.

 

  • Guide scores can vary with cell line, sgRNA and experimental conditions, making it difficult to apply the same counter-measures to every experiment.

 

  • Given multiple cut sites trigger the same effect, how do we ensure multiple sgRNAs when introduced into a cell are not inducing a similar response? This is difficult to control in pooled screens, and poses a limitation in multiplex screens. Synthetic lethality screens for example with sgRNAs targeting multiple genes, might be subject to a higher false positive rate.

 

  • With even diploid genes (copy number = 2) having statistically significant growth reduction compared to haploid gene loci, the challenge still remains to delineate a true loss-of-function over a non-specific cellular response.

 

  • Negative sgRNA controls have to be carefully selected. From the study, non-targeting controls had little impact on viability compared to most other sgRNAs. Controls targeting non-expressed genes or non-essential loci have been recommended as better controls.

 

  • Lastly, although this effect seems to apply mostly to cancer cell lines that undergo a high rate of gene amplifications, similar effects may extend to polyploid tissues such as the liver.

Hence as always gene function should be determined by a variety of methods. Using RNAi for example to affirm a CRISPR-knockout phenotype would add greater confidence to a hit. To avoid those RNAi-related false positives however, its probably best to use siPOOLs.

 

Source of figures:

Aguirre, A. J., Meyers, R. M., Weir, B. A., Vazquez, F., Zhang, C.-Z., Ben-David, U., … Hahn, W. C. (2016). Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting. Cancer Discovery, 6(8), 914 LP-929.

Meyers, R. M., Bryan, J. G., McFarland, J. M., Weir, B. A., Sizemore, A. E., Xu, H., … Tsherniak, A. (2017). Computational correction of copy-number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells. bioRxiv. Retrieved from http://biorxiv.org/content/early/2017/07/10/160861.abstract

Other relevant sources:

Munoz, D. M., Cassiani, P. J., Li, L., Billy, E., Korn, J. M., Jones, M. D., … Schlabach, M. R. (2016). CRISPR Screens Provide a Comprehensive Assessment of Cancer Vulnerabilities but Generate False-Positive Hits for Highly Amplified Genomic Regions. Cancer Discovery, 6(8), 900 LP-913. Retrieved from http://cancerdiscovery.aacrjournals.org/content/6/8/900.abstract

de Weck, A., Golji, J., Jones, M. D., Korn, J. M., Billy, E., McDonald, E. R., … Kauffmann, A. (2017). Correction of copy number induced false positives in CRISPR screens. bioRxiv. Retrieved from http://biorxiv.org/content/early/2017/06/23/151985.abstract

 

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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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