ZEBRAFISH AS IN VIVO MODEL, MICROINJECTION AND CRISPR: THE THREAD

Zebrafish is a well-known model due to its easy study. It has a short life cycle which allow us to obtain information of our experiments within few hours. After one hour from fertilization, the embryo has already 4 cells. In just 72 hours, zebrafish reaches its larval stage.

In the pictures below, I show you different embryonic stages I could take in the lab.

One of the advantages of zebrafish eggs is their availability for microinjection. We can inject mRNA to be translated in the first hours of development as if it were a maternal messenger. We can also inject CRISPR systems. To do this, we first remove the chorion with pronase.

We first need to calibrate the needle for the microinjection. In order to do this, the needle must be cut until a bubble with a size between the two marks is expelled as shown in the picture.

Once the needle is calibrated, the egg is carefully injected. Ideally, the cell should be injected and not the yolk. At the end of an injection row, some eggs may have 2 cells. In this case, both cells are injected.

One way to learn how to inject and check the result easily is to introduce dsRED mRNA (red fluorescent protein). If the mRNA has been injected correctly, red fluorescence can be observed in the embryo after 4-6 hours of development due to expression from the cell.

Once we have experience injecting, we could start using some CRISPR-Cas13d systems. Cas13d is a “molecular scissor” which cuts RNA based on the level of complementarity with a guide (gRNA). Thus, we need to inject the Cas13d mRNA or directly the protein, and the gRNA chosen.

For example, we could introduce Cas13d mRNA plus a gRNA specific for a mRNA involved in tail formation (tbxta gene). The Cas13d protein will be traduced and paired with the gRNA. Then the tbxta mRNA will be depleted and a no-tail fenotype will be observed. WT left, NTL right.

I would like to end the thread showing you the zebrafish mating, which seems like a snowfall to me. Yes, those snowflakes are the zebrafish eggs (some of them fertilized, others not).

I hope you enjoyed it! :)

Please keep going down to see the acknowledgements!

I had the grateful chance to take all those pictures and videos in the @crisprscan ‘s lab. I’ve been learning a lot these weeks there in a good work environment. I would like to thank all the team for helping me everytime, and I highly recommend to take a look at their research!