Snail coiling: CRISPR editing of a single gene turns
righties into lefties
Reiko Kuroda
Frontier Research Institute, Chubu University, Japanrkuroda@isc.chubu.ac.jp
Synopsis

The establishment of left-right (l-r) body asymmetry is a key biologicalprocess which is strictly regulated genetically. We have demonstrated
unequivocally that the handedness of the freshwater snail Lymnaea (L.) stagnalis isdetermined by the single maternal diaphanous gene Lsdia1. The gene operates at the one-cellstage to set the chirality and dictates the entire chiromorphogenesis programme across thelevels of biological hierarchy. We have also revealed that mechanical micromanipulation
during the 3rd cleavage to reverse the spiral-cleavage direction creates mirror-image animals.
Abstract

Most animals display external bilateral symmetry, but the structure and position of thevisceral organs show asymmetries that are determined genetically. To reveal the still
unknown mechanism of the establishment of left-right body asymmetry, we have chosen towork on the freshwater snail L. stagnalis, which has several unique characteristics. 1) Boththe dextral and the sinistral strains within a species are found in the wild with 98% of thesnails dextral and 2% sinistral. 2) The asymmetry is displayed both externally and internally.
3) Snail handedness depends on the direction of spiral cleavages of the embryos, as wasreported as early as 1894.1) 4) The snail is a hermaphrodite, and hence both self-crossing andcrossing can occur. This makes the genetics more versatile and informative.
Fig. 1 shows three left-right pairs of L. stagnalis: a) a pair in the wild, b) one created bymechanical manipulation and c) the sinistral one is created by CRISPR/Cas9 genome editingof the dextral one. Contrary to what has been believed, temporal and spatial cytoskeletal
dynamics for the left- and right-handed snails within a species are not mirror images of eachother. Thus, during the third cleavage, helical spindle inclination (SI) and spiral blastomere
deformation (SD) are observed only in the dominant dextral embryos at metaphase-anaphase,whereas in the recessive sinistral embryos, helicity emerges during the furrow ingression(Fig. 2)2). We have constructed the congenic F10 strain that on average has inherited 99.9%of the sinistral-derived genome and only 0.1% of the dextral strain-derived genome. All thedextral embryos oviposited by F10 animals that inherited the dextrality gene(s) within the0.1% of the dextral-derived genome exhibited dextrotropical rotation with SD/SI at the 3rdcleavage and without exception grew to possess dextral body shape. Equally, all the sinistralembryos oviposited by F10 animals that do not inherit the dextrality-determining gene(s)showed levotropical rotation without SD/SI, and grew to possess the sinistral body shape,again without exception. These findings strongly suggested that dextrality is determined by asingle gene or by genes at closely linked loci.3)
We carried out positional cloning and identified that Lsdia1 is the strongest candidate genefor the handedness determination. There are tandemly duplicated Lsdia1 and Lsdia2 genes
whose proteins share 89.4% amino acid similarity. In the sinistral strains, both Lsdia1 alleles
had a frameshift mutation early in the coding region (c.184delC) that leads to proteintruncation.4) Further, knocking out the Lsdia1 gene using CRISPR/Cas9 produced sinistrallycoiled offspring generation-after-generation in the otherwise totally-dextral genetic
background, if biallelic frameshift mutations had occurred (Fig. 1c).5) We could show that thegene sets the chirality at the one-cell stage, the earliest observed symmetry-breaking event
linked to body-handedness in the animal kingdom. The gene dictates the entire
chiromorphogenesis programme across the levels of biological hierarchy wherein the early
intra-cellular chirality is superseded by the inter-cellular chirality during the 3rd cleavage,
leading to asymmetric nodal/Pitx expressions and then to organismal body handedness.5)
In parallel, we revealed that mechanical micromanipulation during the 3rd cleavage toreverse the spiral cleavage direction creates mirror-image animals (Fig. 1b, 2). The
expression site of the nodal/Pitx genes was inverted by mechanical micromanipulation. (Fig.
2).6)
Diaphanous genes are present in all the Eukaryotes, and therefore, our work featuringformin-controlled early onset of chirality in L. stagnalis may provide new insight on unifying
mechanisms of l-r body plan formation in animals.
a) Sinistral andDextral in naturec) Sinistral created byb) Created byCRISPR/Cas9 from Dextral
micromanipulation

NatureNature

2004 journal cover

2009

2019 journal coverFig.1 Three l-r pairs in the wild (a) and created (b, c).
Offspring reverts
to the originalchirality

References

Fig.2. Breaking of mirror-image relationship,and l-r inversion by mechanical manipulation.

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4. Kuroda, R. et. al., Scientific Reports, 6, 34809 (2016).
5. Abe, M. & Kuroda, R., Development,146 Dev175976 (2019).
6. Kuroda, R. et al., Nature 462, 790-794 (2009).