Month: December 2015

Seed effects persist in hyperdimensional space

Seed effects persist in hyperdimensional space

Work from the Carpenter lab suggests that attempts to shake seed-based off-targets by going to  ‘phenotypic hyperspace’ will not work.

They performed a high-content assay with 315 shRNAs covering 41 genes.  A 1301-dimensional profile was created for each well, and compressed to 205 principal components that captured  99% of the variance.

The hope would be that by examining a wider phenotypic space, the gene-specific effects of RNAi reagents would become more prominent.

However, the profiles between shRNAs targeting the same gene are only slightly better than those between random shRNAs, while shRNAs sharing the same seed sequence have much more similar profiles.

Screen Shot 2015-12-15 at 14.27.51

(figure shows percent of significant profile correlations for different pairings)

Off-target phenotypes can only be escaped by using a reagent that exclusively knocks down the target gene.

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Knocking out the phenotype

Knocking out the phenotype

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 of interest, it is currently very difficult to be certain that this leads to loss-of-function, and hence to be confident about the role of the gene in embryonic development.

Using a knock-down reagent that prevents off-target effects is the best way to be confident about your phenotypes.

 

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