Hi all,
I am looking for a few genes that don’t have a noticeable behavioral phenotype. Ones that are redundant maybe or function in non-essential processes. I have a few guanylyl cyclases to try. Does anyone have any other ideas?
Thanks!
I don’t think no obvious behavioural phenotype = ‘useless’ :o
What are you trying to study? If we have more insight as to what your research question is, we might be able to help you better.
Welcome to the forum…Parasites are worms (shouldn’t that be worms are sometimes parasites? Ed.)
First, as it is Tuesday, a quote from Daniel Day Lewis (aka Abraham Lincoln) :
‘Give me six hours to chop down a tree and I will spend the first four sharpening the axe.’
A well formulated question is likely to get the response it deserves and vice versa. You should perhaps look here:
http://www.pnas.org/content/94/7/3384.full.pdf+html
before committing the guanylyl cyclases to the evolutionary rubbish dump and perhaps here:
http://www.wormbook.org/chapters/www_geneduplication/geneduplication.html
to help formulate your razor sharp follow up post to the forum.
Steve
Okay, apparently my lighthearted post missed the mark. I am looking for a gene to use as a ‘generic’ CRISPR target. Therefore, the gene can not have an essential function, a function in locomotion nor in the process I am trying to study. I would like a way to insert reporter constructs directly into the genome using CRISPR with a ‘generic’ target. Then, I only need to change the cargo not the entire targeting construct. Gcy-4 with the defect of inability to chemotax to bromide or iodide might be considered ‘useless’ whereas gcy-33 which senses O2 might not. I was simply interested to find out if anyone else had favorite genes that could be knocked out with no obvious detriment to the worm.
So, to clarify: you want a loss-of-function phenotype for the knock-in event, but you want it to be one that doesn’t interfere with your desired assays, or for that matter with other popular assays?
It’s not hard to come up with some fairly specific chemotaxis receptors, and to declare yourself willing to sacrifice that detection capability as being unrelated to your interests. Still, I wonder how useful you’ll find it to have a recessive phenotype as subtle (and as labor-intensive) as a Chemotaxis defect.
You might consider suppressors of Rubber-band (unc-93 and so forth). The starting phenotype is perhaps slightly inconvenient (they’re healthy enough to propagate well, but I don’t know I’d care to inject them), and as best I’m aware the loss-of-function phenotype is indistinguishable from wild-type.
Why can’t you just put your genes of interest into the a spot in some 3’ or 5’ utr? There are PAMs practically everywhere.
For subtle phenotypes there’s always the Lox-P / Cre-recombinase method to remove your selection markers once you have a strain with a knock-in (see: http://wormcas9hr.weebly.com/protocols.html).
If you chose the same insertion site you’d only need one sgRNA targeting plasmid and your homologous repair template would contain common 5’ and 3’ homologous recombination arms which would be fairly straightforward to insert various constructs into.
MoSCI sites are another (mentioned) alternative.
The whole idea of using a CRISPR/Cas-9 method is so that you end up disrupting a significantly smaller portion of the genome. I understand that 3’ or 5’ insertions may cause some regulatory effects, but I don’t know if it’s a bigger confounder than removing a gene.