Celebrating 11 years of off-target effects

 

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. show that correlation between siRNAs for same gene is near zero, while seed sequences (involved in off-target effects) account for ~50 times more screening variance

• 2013, Hasson et al. find little overlap between hits from a mitophagy assay run in parallel with different siRNA libraries

Wouldn’t it have been a minor miracle if the phenotype from the following transcriptional profile were due to  knockdown of the intended gene? (intended gene: Scyl1, gene actually responsible for phenotype: Mad2L1, source)

We are not saying that siRNA screens are not useful.  There is some signal amongst the off-target noise.  But luck and a lot of work are required.  Among the top genes from the resulting ‘hit’ list, one must hope that a story can be made (TOMM7, a major character in the Hasson paper, was relatively far down the hit list, and its known location in the mitochondrial outer membrane made it more than a lucky guess).

But there is a better way.  By maximising the separation between on-target signal and off-target noise, siPools can provide clearer phenotypes, thereby reducing wasted effort and dependance on luck.

 

Notes:

Birthday cake created using Fig 2b from Jackson et al. (heat map showing deregulation of off-target transcripts by siRNAs against IGF1R).

Calculation of variance explained by genes vs. seeds (from Marine et al.):

by-gene R = .073; by-seed R = .53; .53 ^ 2 / .073 ^ 2 = 52.71