#disso update

[acuityinnovations]

I had a dream I wrote a poem about zebrafish used as disease models so I made it real. The screenshots throughout are taken directly from my dissertation.

[acuityinnovations]

Want to explain zebrafish genetic expressions?

YOU HAVE OPENED THE FLOODGATES

*flaps hands* yes yes yes ok ok So

In genetics, we have what are called "model organisms" that we run testing on due to their genome's similarity to the human genome, as well as other factors such as quick development and ease of genetic manipulation. One of these model organisms is Danio rerio, the zebrafish. Their embryos are extremely easy to genetically modify, and develop into independence in five days. Full maturation takes approximately three months.

Genes that function identically in two organisms are considered to be "orthologues" of each other. The zebrafish genome has been fully sequenced and is very well understood, with about 70% of human protein-coding genes having zebrafish orthologues, and about 84% of human disease-causing genes being found in zebrafish.

Genetic research into the effects of gene expression and mutation often happens using zebrafish, as we have an array of techniques for things like reducing/enhancing expression, deleting genes, and inserting genes from other species (transgenics). The very first GloFish that were made by splicing the gene for a jellyfish's bright green fluorescence into aquarium fish were actually zebrafish!

My (✨award-winning✨) dissertation (which I never Ever shut the fuck up about, you can find me liveblogging it under the tag #disso update) involved investigating a particular gene in zebrafish called nat15, which in humans is called NAA60 (N-alpha-acetyltransferase 60). These two genes have the same function, which means they are "orthologues" of each other. This function is to modify proteins as they are being made.

We investigated whether dysfunction of this gene causes neuromuscular symptoms to develop. In doing this we caused deletion of nat15 in the zebrafish embryos using the CRISPR/Cas9 gene editing system, and found that it caused them to move slower, move less, respond less to stimulus, and lose muscle integrity. They also displayed microphthalmia (abnormally small eyes) and cardiac oedema (fluid buildup in the heart).

CRISPR/Cas9 consists of two parts:

- Cas9 endonuclease that "cuts" the DNA sequence

- Single-guide RNA (sgRNA) that tells the Cas9 where to cut

RNA is single-stranded instead of double-stranded like DNA. sgRNA itself is made of a further two parts:

- CRISPR RNA (crRNA), which has a "complementary" matching sequence to the DNA you want to cut

- Trans-activating CRISPR RNA (tracrRNA), which provides stability.

CRISPR/Cas9, when used to delete a gene in what we call gene knockout, happens like this.

1. The sgRNA finds the gene of interest with its complementary sequence.

2. The Cas9 makes lots of cuts in the gene called double-stranded breaks (this means that both strands of the DNA are cut - if it were just one strand we call that a "nick" and we use Cas9 "nickase" instead of Cas9 "nuclease").

3. The DNA sequence then attempts to repair itself in a process called "non-homologous end joining". The DNA joins itself back up, but because there are so many breaks, it makes lots of errors and the original sequence is ruined.

4. Because the original gene sequence is now unrecognisable, the gene can't be read and does not function. It is now "knocked out".

We did this to nat15 in our zebrafish embryos, and found that some of the symptoms produced were similar to those found in the human disease "primary familial brain calcification" (PFBC) - a disease primarily marked by calcium deposits in the brain. This means that we can further investigate NAA60 in humans as a causal gene for PFBC! My dissertation supervisor is currently furthering my research and creating a stable mutant line of nat15-mutated zebrafish, which means that the mutation can be passed on reliably through each successive generation.

In conclusion, zebrafish are cool as fuck, genetics is cool as fuck, and thank you for giving me the opportunity to infodump :3 If you wanna read my dissertation in full I'd be happy to send it to you!!

[acuityinnovations]

I SUBMITTED MY DISSERTATION!!!!

IT'S DONE I'M SO FUCKING HAPPY AAAAGH!!!

[acuityinnovations]

dissertation update:

THE DRAFT IS DONE AND SUBMITTED!!! EIGHT STRAIGHT HOURS OF OVERNIGHT WORK I AM THE LITERAL KING OF SPEEDRUNNERS

got 4% on my turnitin report too :D

[acuityinnovations]

dissertation update:

DRAFT IS DUE IN 14 HOURS AND I AM NOT FINISHED OUUGHH

pray for me y'all its 2am and i'm in uni finishing it

[acuityinnovations]

dissertation update: wrote 800 words today! methods section done :3

we were supposed to do tubulin staining on our embryos to check neurogenesis in the crispants but my supervisor just straight up forgot 🙃 AND he did some extra rounds of injections at a lower dose and they turned out with brand new symptoms and he just....didn't tell me....

1 like = 1 time i hit him on the head with a baseball bat

[acuityinnovations]

dissertation research update:

lab based research is finished! i am, actually quite upset about it :( i was having so much fun and my supervisor was so friendly!! i got him a little parting gift (some fancy cheese) and he was DELIGHTED

he was in a meeting when i finished up in the lab so i sent him an email, and his reply was so sweet 🥺 especially this part

don't mind me, i'm ugly crying.

so labs are finished and all i have to do is write it up, y'all are free to message me about the deets! it was a privilege to work on this project and to have such a cool supervisor :) cheers dan, you're the best!!

[acuityinnovations]

dissertation research update: ok so. using all 3 cutting guides at once for a 100% single gene knockout in 24hpf zebrafish embryos is. Not Viable

on one hand, poor fish :( (it's ok they didn't feel anything, they were hardly past being zygotes and were still in their chorions). on the other hand, an interesting discovery! interesting implications for the role of our gene of interest in early cell development 👍🏽

we're going to try again next week with a lower dosage to check if it's a dosing issue or not