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

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

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.

Single-cell embryos were microinjected with CRISPR reagents. Embryos were held in a 3D-printed agarose dish, and dextran dye confirmed injections.

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.

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.

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

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