Gene editing without a genome: generation and validation of F0 CRISPR mutants in gastropod mollusc Crepidula fornicata

Background CRISPR-Cas9 gene editing is a powerful tool to study gene function but to date is mostly applied in traditional model organisms. Due to specific challenges in spiralian genomics such as genome size, complexity, proportion of repetitive regions and high inter individual genome variation, applications of CRISPR-Cas9 in spiralian models have been limited. Within the Spiralia, molluscs are a strikingly diverse phylum with many unique gene family expansions and novelties, yet there are relatively few applications of CRISPR-Cas9 to unravel gene function. Results We generated pax6 knockout F0 CRISPR-Cas9 mutants in the gastropod mollusc Crepidula fornicata using a de novo transcriptome to design single guide RNAs and genotyping primers. In lieu of an assembled genome, alignments with closely related species were used to determine putative intron-exon boundaries and successfully target gene editing to a specific exon of pax6 containing a homeodomain. F0 pax6 knockout mutants had an eye-loss phenotype. Successful CRISPR-Cas9 gene editing was confirmed genotypically using Sanger sequencing and Interference of CRISPR Edits (ICE) analysis. Conclusions The generation of F0 CRISPR-Cas9 knockout mutants with a clear phenotype in a mollusc without an assembled genome enhances the applicability of CRISPR for functional genomics in non-traditional emerging model systems. pax6 is highly conserved and is required for eye development across metazoans, including the snail C. fornicata . The pax6 mutants developed in this study suggests the pax6 homeodomain is specifically required for eye formation in gastropods and sheds light on the evolution of eye development in animals.