Progress on my project has unfortunately been quite slow, due mostly to unavoidable issues that cropped up earlier into the process. In particular, the change from using FL green sea urchins to NC green sea urchins. Given that the project started originally with using the genomic information gathered from FL urchins and the vast genetic variation between population of sea urchins, the guide RNA created for the FL urchins, while successful on those urchins, has proven to be little effective on the NC variety. The reason for the switch from FL to NC urchins is the season in which the animals are reproductively active and thus when their eggs are most healthy for injections.
However, with the lack of genomic sequencing of NC-originating Lytechinus urchins, it has been impossible to create gRNA specifically for the urchins we are currently using. Luckily, one of the members in the lab has recently been sequencing some sections of the NC sea urchin genome, so we may soon be able to design new guides that have a higher effectiveness, assuming that section of the genome has been sequenced and the gRNAs initially created are indeed different enough to warrant new guides.
On a brighter note, I’ve been practicing injections most days of the week and have been able to better hone my skills on the practice as it can often take months to learn how to inject efficiently. Early on, I would often take up to an hour trying to inject a single plate of embryos and awhile to setup my materials for the day. Now, I’ve managed to better refine the protocol and can have everything ready in an hour and do one plate every 20 minutes. Though that’s still a bit long (the optimum time being 15 minutes) and I still need to work on injecting more embryos in that time frame, I feel as though I’ve improved quite a lot and learned a number of things in the process.
Abstract- What Are the Functions of Genes in the GRN of Sea Urchins?
Sea urchins are one of the most useful model organisms for studying early development; however, much is still unknown about the specific functions of genes within the network that regulates urchin development. One gene that has been well-studied is Endo16 and its importance in gut formation. Using CRISPR/Cas9 technology, this project has focused on producing successful knockouts of Endo16 by targeting the gene’s enhancer region. Thus far, there has been moderate success with knocking out Endo16 in Lytechinus embryos, though further experiments are needed to improve the success of the Cas9 knockouts by taking into consideration the significant genetic variation between populations of Lytechinus. Once the percentage of embryos that are injected produce Endo16 knockouts equivalent to the percentages seen in similar studies using other gene silencing techniques, the project will move forward with knocking out other genes whose functions are less well-understood compared to Endo16. This will further our understanding of the gene functions and relationships within the gene regulatory network of sea urchins, and allow comparative studies of gene function in Lytechinus and the closely-related, yet developmentally very different, species, Heliocidaris erythrogramma.