Germline Editing in Marsupials Marks Another Milestone for CRISPR

CRISPR-based gene editing has slowly and steadily made its way into tests in cell lines, plants, mice, lizards, squids, and humans. It is a mainstay in research and lately approved for clinical therapy for editing somatic cells. However, researchers are extremely cautious about germline editing due to the challenges in off-target effects, editing efficiency, and unforeseen mutations. 

In a new study published in Current Biology, researchers have demonstrated successful germline CRISPR editing in opossums. Opossums belong to the mammalian subfamily of marsupials, like kangaroos, wallabies, and koalas. They are model organisms for reproductive biology research since much of their early development occurs in the external environment. For the same reason, CRISPR-editing the marsupial genome is tricky.

Study Design

In a controlled study, scientists at the RIKEN Institute, Japan, knocked out the Tyrosinase (Tyr) gene for pigment production in the gray, short-tailed opossum. Successful homozygous knockout could be easily visualized by albino progeny, and heterozygous deletion would result in mosaics.

While the CRISPR design was fairly straightforward, the authors had to improve methods, the timing of delivery of guideRNA into the embryos, and transfer of edited embryos into pseudopregnant females. They had to navigate the hard-to-penetrate mucoid shell, covering the fertilized eggs.

Using a piezoelectric needle, they were able to safely inject the embryos at the pronuclear stage. The first litter contained one albino and one mosaic opossum, demonstrating successful homozygous and heterozygous deletion by CRISPR. Breeding the founders into the next generation resulting in an all-albino litter, indicating that the editing is inherited and maintained in the germline. 

Significance of the Study

This study can help comparative biologists to understand the unique aspects of marsupial and non-primate biology by reverse genetics – systematic deletion of genes to study their function. Genes that are conserved in all mammals are probably essential with a similar function, and those that are unique can help understand the differences. Marsupials are special in their

1. Reproductive and development biology: opossums are born as a ball of cells with forelimbs after a short gestation of 14 days. Much of the development takes place in the marsupial membrane attached to the mother. Hence, they are an ideal system to understand the early development of mammals. 
2. Immunology: marsupials contain three classes of T-cells, compared to two isoforms in most vertebrates. Early development in the external environment may also require a robust and sophisticated immune cell network. Knockouts can help us understand these unique parts of the marsupial immune system.
3. Ability to develop melanoma: Opossums can get skin cancer and are model organisms for research combining melanoma and immune response to develop new therapies.

Successful germline editing paves the way for developing therapies for congenital defects, monogenic diseases such as muscular dystrophy, and reversing the effects of pathogenic mutations in hereditary disorders. It encourages stakeholders to develop a regulatory framework for evaluating the cost-benefit analysis of germline editing.

Author

Sahana Shankar