The pax6 homeodomain eyeless KOs confirms the conserved role of pax6 in eye development in molluscs. We think C. fornicata could be a useful, genetically tractable system for visual system evolution studies in addition to the fabulous apple snail model developed by @accorsi-alice.bsky.social
So, for the embryo and developmental biology lovers out there, transcriptome-guided CRISPR can produce useful F0 knockouts with a clear phenotype, enabling gene function studies in non-traditional emerging model systems. #CRISPR #GeneEditing #ModelOrganisms #DevBio #Transcriptomics
Live microscopy images of Crepidula fornicata early veliger embryos. One has two eyes, one has no eyes as a result of a pax6 knockout.
Eye phenotypes were dramatic: embryos developed two, one, or no eyes. Disruption of the pax6 homeodomain inhibitted eye formation, confirming its highly conserved developmental role. #LookMaNoEyes
Chromatograms of wild-type, single guide RNA injected with no cas9, and F0 mutant pax6 Crepidula fornicata samples.
Genotyping revealed mosaic F0 mutants. Multiple alleles, including in-frame and out-of-frame mutations, were present at the sgRNA target site, while controls remained unmodified.
A figure showing a 3D printed mould that is stamped into agarose to make angled microinjection wells for embryos.
Single-cell embryos were microinjected with CRISPR reagents. Embryos were held in a 3D-printed agarose dish, and dextran dye confirmed injections.
A schematic alignment between Littorina saxatilis pax6 and a Crepidula fornicata pax6 partial transcript.
We identified a single pax6 transcript in the transcriptome, a master regulator of eye development. Alignments with other gastropod genomes revealed putative exon-intron structure, allowing exon-specific primer and sgRNA designed to target the homeodomain.
H&E staining on sectioned Crepidula fornicata eyes throughout development.
In C. fornicata larval eyes start as simple pigmented cups from ectodermal invagination, gaining pigment and a tiny lens by the veliger stage. Adults have lenticular eyes with a cornea, acellular lens, dense pigment, and layered photoreceptors.
A DAPI stained early veliger Crepidula fornicata embryo from the left lateral view.
CRISPR in molluscs is challenging: delivery is hard, embryo husbandry is hard, genomes are huge and complex. We tested whether efficient F0 knockouts could be generated in the slipper snail, Crepidula fornicata, using only a transcriptome to guide us. @vs-marine.bsky.social
Happy holidays to one and all ❄️! On the Friday before Christmas 🎄 I’m excited to share my first first-author pre-print. Are you interested in molluscs 🐚, developmental biology, CRISPR F0 KOs, Pax6, eye 👁️ development and evolution? Then here’s a thread for you. www.biorxiv.org/content/10.1...
PhDone ✅🐚✨
For today's quick sketches I got to draw sonic the hedgehog (which is a fabulous gene name)
First preprint from my PhD is out!
If molluscan developmental biology and genetics, gene-editing methodology in non-traditional model systems, or the function and conservation of pax6 interests you, please give it a read! 🐚🎉
Shucked stack of snails (say that five times fast)
Exposed for my frantic 6 hour day of microinjecting crepidula embryos at @mblscience.bsky.social with @vs-marine.bsky.social. Heavy amounts of Ed Sheeran in the lab that day (Unofficial Sleight Lab Work music)
Sorry Aberdeen is BYOVCADWP (bring your own vaulted ceiling and dark wooden panels)
Looking at this note my PI left on a waste bottle and thinking--what could it mean?
Jürgen is all of us entomologists 😂 still love this moment! #gbbo
I'll be at the #MalacologicalSocietyofLondon meeting this Wednesday! If anyone is interested CRISPR/cas9, snails, and cool microscopy, images, come listen to my talk and have a chat with me!
Hello all! Joining Blue sky to engage with the scientific community and to talk about #snails follow for updates on my research and science-related shenanigans!
