siTOOLs Biotech Blog

Consistent with the work of Rossi et al. (discussed previously), another recent paper shows a lack of phenotypic response when knocking out a gene that gives a phenotypic response when knocked down. Knocking out klf2a does not result in any discernible difference from wild-type (whereas knock-down has been shown to produce a range of cardiovascular phenotypes). The authors conclude: In summary, our work shows that even in the face of clear evidence of a potentially disruptive mutation induced in a gene Read More

Genetic compensation

25. September 2015 Andrew Walsh

Recent work by Rossi et al. shows that an unintended consequence of gene knockout may be genetic compensation that mitigates phenotypes. Knockdown in zebrafish of egfl7, an endothelial extracellular gene, causes severe vascular defects: However, following knockout of eglf7, there was no visible effect on vascular development, even after application of the knockdown reagent (demonstrating that the knockdown phenotype was not due to an off-target and that the knockout’s normal vascular development was not due some minor levels of egfl7): The authors found Read More

Russian roulette

19. February 2015 Andrew Walsh

Which bases should you choose for the seed region of a single siRNA? It’s like Russian Roulette on full-automatic, where a specific seed will result in dozens or hundreds of down-regulated genes. If you’re lucky, none of the off-targets results in a false-positive phenotype. But odds are that you won’t be so lucky. A recent paper suggests that taking single siRNA drugs may be closer to real Russian Roulette than anyone would hope. The authors show that an siRNA designed to knock down Read More

The beauty of the siPool

22. October 2014 Andrew Walsh

The siPool strategy is beautifully simple: By having many on-target siRNAs, each with a different seed sequence, you maintain on-target efficiency while diluting out off-target effects. One analogy is the beauty of composite faces. Which of these faces do you find most attractive? If you’re like most people, you will have chosen the last face, which is actually a composite of the other 5 faces (source). Each individual face has its flaw(s). Spock ears, mildly everted lips, incongruous eyebrows, etc. No Read More

The nasty, ugly fact of off-target effects

15. October 2014 Andrew Walsh

Once upon a time, it was imagined that siRNAs specifically knock down the intended target gene. Unfortunately, this turned out to be wrong. The disappointing results from siRNA screening following the initial high hopes brings to mind T.H. Huxley‘s famous quote about a beautiful theory being killed by an ugly, nasty little fact. As pointed out by S.J. Gould in Eight Little Pigs, the origin of the quote is given in the autobiography of Sir Francis Galton. Galton’s autobiography is inspirational. Read More

Don’t swallow the fly

9. October 2014 Andrew Walsh

There was a PI who screened one s i [RNA], Oh, I don’t know why they screened one s i … siRNA screens have a high false positive rate, due to pervasive off-target effects. Confirming ‘hits’ from single-siRNA screens is a lot of work. For low-complexity pool screens, it’s even worse (and, as we will discuss in a later post, less likely to result in true genes of interest). Progressively, one accumulates a nearly indigestible set of experiments and analyses. Read More

Celebrating 11 years of off-target effects

2. October 2014 Andrew Walsh

This year marks the 11th anniversary of Jackson et al.‘s seminal paper on siRNA off-target effects. The past decade of high-throughput siRNA screening is largely a deductive footnote to their observation that “…the vast majority of the transcript expression patterns were siRNA-specific rather than target-specific“. 2005, Lin et al. show that top hits from RNAi screen are due to off-target effects 2009, Bushman et al. report poor overlap between hits from HIV host factor screens 2012, Marine et al. Read More