Auxin-induced degradation in C. elegans

Model Organism: Worms Scientific Discussion
1

Hello everyone!

I am designing my CRISPR insertion of the degron sequence in a specific gene in order to do auxin-induced degradation in C. elegans. I was wondering if anyone can help me out!

  • We will insert Degron::emGFP sequence in our gene of interest by CRISPR. Should we add a linker?

  • How to decide where to place degron, whether 5’ or 3’ of the gene? Depending on the domains of the protein of targeted? Or do you suggest putting the degron::emGFP sequence 5’ of our gene of interest?

Any information is useful! Thank you!
Victoria Cerdeira

2

I think that there’s not really a universal rule, it really depends on the gene. In our typical experiments we:
-try to label all isoforms, which affects whether we do N-terminal, C-terminal, or internal tagging (later we may try isoform-specific AID-tags)
-if the gene isoforms don’t dictate tag placement, we try to place the tag away from protein domains, in case the insertion affects protein function
-we use a linker to separate the FP::AID cassette away from the protein

One other consideration is your CRISPR approach. We use SEC selection frequently, so N-terminal tagging produces a promoter reporter and inactivates the gene in the initial insertion and a translational fusion after cassette excision. This feature is very nice, though if it’s an essential gene will require that you balance the insertion in order to excise the SEC (which is more work).

Cheers,

Jordan

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3

Hi Vicky -

Including a small linker (e.g., GGSG) does not usually cause any problems and can prevent them. That said, the degron is itself a fairly unstructured, flexible peptide so it may not be necessary to add a linker between your protein and the degron - it’s more likely to help between your protein and GFP.

As with epitope tags, the optimal insertion point for the degron should be determined by educated guessing and experimentation. Different people use different rules of thumb. My own approach is to quickly determine which part(s) of the protein are most highly conserved by downloading the Caenorhabditis orthologs at http://download.caenorhabditis.org/v1/json/CGP_orthology.html and generating a multiple sequence alignment using MAFFT (you can also download a pre-calculated MSA at that site). All things being equal, I would usually choose to tag the less conserved end (unless, for example, it’s only expressed in a subset of isoforms). If both ends are strongly conserved or predicted to be important, an internal insertion into a flexible loop may be worth testing.

Also, we recommend editing directly in strains expressing the TIR1 construct you plan to use - it’s simple and allows you to test the efficacy of your degron construct without crossing.

Good luck! If you have any other questions about this, I’m happy to try to address them.

Abby

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4

Hello, I am very interested to try the Auxin-induced degradation in C. elegans. I am very new to this technique and wondering if anyone could direct us to resources on how to design construct and insert degron from the very beginning?

Thank you very much.

Kin