In a recent paper in Current Biology, IGTRCN Participant Arnaud Martin and colleagues use Cas9 gene editing technology to study the development of the crustacean Parhyale hawaiensis. In this study by Martin et al., the specific morphology of serially homologous appendages is examined by applying CRISPR/Cas9 to several Hox genes.
While CRISPR/Cas9 mutagenesis and editing are often employed in the germline to create vertically transmitted changes in the genome and permanent lines of genetically modified organisms, deploying these technologies to just the soma can also be a highly effective approach. Martin et al. target Cas9-mediated mutations to the somatic cells of Parhyale hawaiensis, an amphipod crustacean currently emerging as a model organism. Using CRISPR/Cas9 in this species was relatively inexpensive, at $80 in reagents per target, and samples can be prepared in two days. To rule out off-target effects and to test reproducibility, the authors targeted several different clusters of nucleotides resulting in the same mutant phenotypes for each sgRNA.
Somatic mutagenesis was achieved by performing injections of Cas9 mRNA and sgRNA into zygotic somatic cells. In this case, the authors injected one of two cells that arose after the first cleavage event following fertilization. This first division determines the fate of each cell to forming structures on the left or right sides of the developing body plan. By injecting only one cell, wild-type gene expression levels occur in one half of the developing embryo but not in the other. This is highly advantagous because one half of the animal serves as an internal control; comparing unilateral mutant phenotypes to wild type phenotypes within the same animal eliminates uncertainty arising from variation amongst individuals. This method can also determine phenotypic intermediates by generating mosaic individuals wherein large clones of bi-allelic knockouts result in reduced levels of expression on the side arising from the injected cell.
Loss-of-function experiments were performed on 6 Hox genes: Deformed (Dfd), Sex comb reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B). Five out of the six showed 25-70% penetrance, supporting CRISPR as far more effective than RNAi. The authors, however, assert that the combination of CRISPR and RNAi approaches was most valuable in defining each appendage’s relative dosage sensitivity to Hox gene expression levels.
Overall, the authors hope that the usage of CRISPR technology for somatic mutations will contribute gene function analyses to expand our current understanding of segmental and serial homolog evolution.
Martin, Arnaud et al. (2015) CRISPR/Cas9 Mutagenesis Reveals Versatile Roles of Hox Genes in Crustacean Limb Specification and Evolution Current Biology , In Press Corrected Proof DOI: http://dx.doi.org/10.1016/j.cub.2015.11.021