RNAi off-target effects takes an interesting turn for cancer research as reported in eLIFE by Putzbach et. al. A specific group of survival genes in cancer cells was revealed thanks to the off-target effects of siRNAs/shRNAs.
The most common way to validate hits from Dharmacon siGENOME screens is to test the individual siRNAs from candidate pool hits (siGENOME reagents are low-complexity pools of 4 siRNAs). In this deconvolution round, we normally see that the individual siRNAs for genes behave very differently and seed effects dominate (discussed here and here). One could argue that deconvolution is not the correct way to validate candidate hits (even though it’s the method recommended by Dharmacon), as testing the siRNAs individually Read More
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 Read More
Genes hardly ever work alone, functioning instead in complex gene networks. Increasing advances in genomics and proteomics and corresponding developments in computational analysis, has really put this into perspective. A recent large scale RNAi study by Novartis found this hairball of a gene network in cancer cells: A gene “hairball” As such, the standard approach of disrupting the expression of a single gene to study loss-of-function phenotypes may not accurately reveal its genetic function. The highly redundant nature of signalling pathways Read More
Pleasantville is a movie based on an interesting idea: two teenagers are magically transported through their TV to a town called Pleasantville set in the 1950s where everything is perfect (and also black-and-white). As they discover the complex, imperfect emotions hidden below the idyllic surface, the black-and-white characters and objects start to gain colour. In loss-of-function genetic screening, some reagents and screening formats may also give rise to a narrow, black-and-white view of a biological process. A sort of “Phenoville”. This was illustrated nicely Read More
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 Read More
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 Read More
You might have heard or participated in the global discussion over the recently published Nature Commentary that described >1000 off-target mutations in CRISPR-edited mice. The paper reported a small study involving three mice but gained enough virality online to trigger a significant drop in share prices of companies founded based on CRISPR gene-editing – Editas Medicine, CRISPR Therapeutics and Intellia Therapeutics. Here is a summary of the study, with respective concerns raised by the scientific community regarding the validity of Read More
As discussed previously, deconvoluted Dharmacon siGENOME pools often give surprising results. (Deconvolution is the process of testing the 4 siRNAs in a pool individually. This is usually done in the validation phase of siRNA screens.) One way to compare the relative contribution of target gene and off-target effects is to calculate the correlation between reagents having the same target gene or the same seed sequence. One of the first things we do when analysing single siRNA screens is to calculate a Read More
The reproducibility of different CRISPR or RNAi reagents targeting the same gene is sometimes cited as prima facie evidence for the superiority of CRISPR screens to RNAi screens. A landmark paper by Shalem et al. showed that different gRNAs inhibit gene expression much more consistently than do different shRNAs: But does this ensure that CRISPR screens are more reliable (as determined by assay reproducibility) than RNAi screens? Not necessarily. Shalem et al. performed two pooled CRISPR screens in parallel, and found substantial overlap between the top Read More
