Hello everyone
Has anyone tried feeding RNAi using short dsRNA, like <50 bp? I’m trying to target circRNAs and only the sequence in the junction is kind of unique.
So I’m wondering if only several tens bp of dsRNA is efficient enough to induce RNAi by feeding.
Thank you.
Hello
Interesting question. I tried to answering using public data.
The sjj clones are all of a similar size, but the mv clones have inserts up to >8kb.
There are only a few <= 150 bp (information easily pulled from http://bioinformatics.lif.univ-mrs.fr/RNAiMap/getclones.php):
mv_C06E1.5
mv_C06E1.6
mv_C25A8.1
mv_C36A4.10
mv_F30F8.7
mv_F35F10.14
mv_F54D12.4
mv_H39E20.1
mv_R08C7.1
mv_T12D8.5
mv_W05H9.3
mv_W05H9.3
mv_W06A7.5
mv_Y105C5A.10
mv_Y17G9B.2
mv_Y39A3CL.3
mv_Y41E3.5
mv_Y44A6D.1
mv_Y48A6B.4
mv_Y48G9A.a
It used to be that one could input a list of clones into WormMart and get back associated phenotypes.
This may be possible with WormMine, but I haven’t found a way to do it; I’d be interested to learn.
One could search these individually at Wormbase. I haven’t done that, but I can say that we pulled mv_F35F10.14 out of a screen for Nipi genes (https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-016-0256-3).
It has a 150 bp insert. That the shortest I know of.
Hope it helps.
Anectodal evidence has suggested 500 is a lower limit. We attempted to knock down a gene with a very robust phenotype (pha-4, no need for IPTG induction and can be diluted 1:8 and still produce a phenotype in a % of the animals) with a 250bp dsRNA fragment, and it failed. But it could be a context dependent phenomenon, so I can’t say that 50bp wouldn’t work - but there’s no evidence to support that it would.
Hope that helps!
Steve
Hi circR,
be aware that RNAi can spread in the worm, so it might happen that you will knockdown your mRNA as well.
Additionally make sure to have a control where the RNAi machinery is getting activated, to be sure whatever you see is not a side-effect.
Surely Cas13 could be very useful for you, but unfortunately it is not yet published for C. elegans.
Hi Steve,
Thank you for sharing your experience.
Maybe I need to try siRNA injection.
Hi Jonathan
Thank you for giving so much information. It seems that the length requirement is different for different targets.
Do you have any idea about why shRNA or siRNA is not preferred in C. elegans? Is it because it’s much easier to scale up by feeding RNAi?
Thank you.
Hi Jonathan
Thank you for your input and suggestions.
Yes, cas13 may be ideal. Maybe siRNA injection will also work.
You said “RNAi spreading”, do you mean it can “spread” between 2 targets?
If I design a short dsRNA or siRNA specific to the circRNA, theoretically, it should not affect the mRNA, right?
Thank you.
Although “RNAi spreading in the worm” is a phrase I’d normally associate with transport between cells (ie sid-1), in this context it seems to be a reference to RNA-dependent RNA polymerase activity, which has been shown to be able to take dsRNA targeting a message and generate RNAi activity against sequences in that message 5’ of the dsRNA homology (eg Sijen et al).