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Tag: knock-out compensation

Clearly compensating

Clearly compensating

Genetic compensation by transcriptional adaptation is a process whereby knocking out a gene (e.g by CRISPR or TALEN) results in the deregulation of genes that make up for the loss of gene function.

A 2015 study by Rossi et al. (discussed previously) alerted researchers that CRISPR/TALEN knock-out experiments may be subject to such effects.

Genetic adaption or compensation had been well known to mouse researchers creating knock-out lines.  In fact, one of our company founders also ran into this when trying to confirm an RNAi phenotype in a knock-out mouse line.  The knock-out mice, though not completely healthy, did not confirm the RNAi phenotype.

A paper published a couple years before the Rossi paper also showed clearly that knock-outs can create off-target effects via transcriptional adaptation.

Hall et al. showed with an siRNA screen that the centrosomal protein Azi1 was required for ciliogenesis in mouse fibroblasts, confirming previous work in zebrafish and fly.

Their Azi1 siRNA targeted the 3′ UTR, and they were able to rescue the phenotype with a plasmid expressing just the CDS (bar at far right), confirming that their phenotype was due to on-target knockdown:

However, knock-out mouse embryonic fibroblast cells (created by gene trapping) did not show any differences in in the number of cilia, centrosomes, or centrioles compared to wildtype (+/+ is wild type, Gt/Gt is the homozygous knock-out):

The one phenotypic difference they observed was that male knock-out mice were infertile, due to defective formation of sperm flagella.  Female mice had normal fertility.  Both were compensating, but only one showed a visible phenotype.

The authors note the benefits of RNAi in comparison to knock-out screening:

Discrepancies between the phenotypic severity observed with siRNA knock-down versus genetic deletion has previously been attributed to the acute nature of knock-down, allowing less time for compensation to occur

The excitement surrounding CRISPR should not diminish the continued value of RNAi screening.

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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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Genetic compensation

Genetic compensation

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:

Screen Shot 2015-09-25 at 21.44.15

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):

Screen Shot 2015-09-25 at 21.56.06

The authors found that in egfl7 mutants, Emilin genes were upregulated.  Like egfl7, these genes are involved in elastogenesis, and thus their up regulation could be compensating for missing Egfl7.  (It seems that humans are also able to compensate for loss of Egfl7).

Work by Kok et al. also reported discrepancies between the phenotypes elicited by knockdown versus knockout experiments.  They found that the vast majority of phenotypes from knockdown experiments were not confirmed by knockout experiments.  They concluded:

Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos [knockdown reagent] that recapitulate respective phenotypes could be reliably applied for ancillary analyses.

People are understandably wary of knockdown phenotypes, given the prevalence of off-target effects.  But the work by Rossi et al. suggests that gene inactivation may give misleading results about gene function.  The best metric for defining gene function will be gene knockdown experiments using reagents that prevent off-target effects.

 

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