I successfully used crispr to edit mosquitoes!

[mr715]

Next step in my work is establishing strains that will carry the mutation.

We are living in the golden age of biology.

[erronis]

1. My bug zapper does that too!

[mr715]

2. The handheld ones are very satisfying.

[NNadir]

3. Cool. What kind of gene did you insert?

[mr715]

4. Tryptophan hydroxylase.

Not insertion. Knock-out.

Disrupting the serotonin synthesis pathway in the central nervous system. Pure science objective of understanding what serotonin really does. Applied science goal of developing novel methods to decrease mosquito host-seeking to prevent disease transmission.

[NNadir]

5. That my friend, is very cool. I have to say that tryptophan has always been one of my favorite amino acids, probably...

...because of its neurological implications.

(Among other things, I'm an insomniac, so there's that.)

It would not have occurred to me, although it probably should have, that serotonin chemistry is neurologically conserved across animal species, but on reflection, it make sense that it is.

Is the hydroxylase in question fairly well conserved across animal species? It has been my pleasure to select appropriate peptides in cross species gene insertions to avoid conserved sequences.

[mr715]

6. It is amazingly well conserved.

It happens to have a eye catching "HELL" motif in its amino acid sequence that is shared from worms to us.

It is a nifty little enzyme, and serotonin is a cool molecule.

[NNadir]

7. Very cool. By "HELL" you mean histidine-glutamic acid-leucine-leucine or something else?

[mr715]

8. Yeah.

More specifically GHELLGH or something like that. coordinates an iron molecule.

[NNadir]

9. Cool. Do you happen to have the Uniprot accession number? I'd like to look into this one, nonprofessional interest...

...just because, well, just because...

I'm a mass spec kind of person as far as proteomics goes, and I often find myself contemplating sequences across species, as I have been peripherally involved in gene modification for genetic diseases. It comes up when one is inserting a human gene in a monkey to experiment in preclinical trials with treating genetic syndromes.

[mr715]

10. Here is a link to it in uniprot

https://www.uniprot.org/uniprotkb/Q8IWU9/entry

Q8IWU9


This is the human protein, but it shares amazing conservation. I think my form is still in the uncharacterized category but better model organisms share it and are pretty close to the mosquito.

It also happens to be a beautiful, symmetry homotetramer.

A former advisor of mine told me to stay away from serotonin research because it would "make me crazy," which is a spectacular line. I love him.

[NNadir]

11. Very cool. I like your advisor's wit. Into what species of mosquito did you knock out the gene?

Has the mosquito been fully sequenced?

An interesting question is whether the mosquito will be viable without the gene and its translation; might it not starve to death?

[mr715]

12. Yes, it has a fully sequenced genome.

Based on work in Drosophila, the knockout mutants are viable.

This same mutation has been done in mice and they are also viable, just highly anxious and aggressive.

The interesting thing about this particular enzyme is it has two isoforms: one that generates serotonin in peripheral tissues, and one that solely operates in the nervous system. I'm knocking out the nervous system one.

The literature seems to suggest that dopamine circuits compensate for the missing serotonin, and there is enough in the somatic cells to prevent major developmental problems.