CRISPR/Cas9 knockouts invert snail shell coiling
To establish that Lsdia1 is the handedness-determining gene and to elucidate what role Lsdia1 has in the cellular programme that directs snail chirality, we developed Lsdia1-targeted CRISPR/Cas9 techniques (Jinek et al., 2012) to create gene-knockout snails (Fig. 1A,B). Out of four independent injection experiments, we obtained 10 CRISPR/Cas9-injected adult snails (numbered 1-10) and two control adult snails (11-12) (Fig. S1B,C). By using DNA extracted from the clipped foot while keeping the snails alive, heteroduplex mobility assays (HMA) (Zhu et al., 2014) were carried out on the snails (Fig. 1B), which showed mosaic mutational patterns at the F0 stage (Fig. 1C). Germline transmission to F1 embryos occurred in snails 2, 3, 5, 8 and 10, of which snails 2, 3 and 10 produced only dextral offspring, whereas snail 8 had only sinistral offspring (Fig. 1D; Fig. S2; Table S1). Remarkably, snail 5 produced both dextral and sinistral F1 (denoted as 5-F1-dex and 5-F1-sin) within an egg mass (Fig. S2B; Table S1). This must be a consequence of mosaicity of frameshift and non-frameshift mutations introduced into the tissues of the gonad at F0, which are originated from the 4d mesentoblast (Morrill, 1998). When and where in the gonad the Lsdia1 mRNA is transcribed and provided to the oocyte is an interesting unresolved issue.