CRISPR-Cas9 Overcomes Obstacle, Uncovers Path to Marsupial Models

Genome editing has been used to alter the genomes of a number of different creatures, including plants and humans. Now, researchers at the RIKEN Center for Biosystems Dynamics Research (BDR) have successfully proven gene deletion in a marsupial, adding another animal to the list of animals genetically modified using CRISPR-Cas9.

This discovery was published in Current Biology as “CRISPR/Cas9 genome editing for targeted gene disruption in a marsupial, Monodelphis domestica.”

The researchers focused their efforts on a gene involved in the creation of body pigments; when this gene is disrupted, the animal loses its colour. More precisely, the scientists demonstrated “the development of Tyr locus gene knockout opossums using microinjection of pronuclear stage zygotes and germline transmission of the changed alleles to the F1 generation.”

The grey short-tailed opossum (Monodelphis domestica) is one of the few marsupial species with established laboratory models. Puppies are born prematurely due to the opossum’s absence of a functional placenta. When exposed to ultraviolet radiation, the opossum, like humans but not other non-marsupial animals, develops skin cancer. Additionally, newborn opossum pups with spinal cord injury, unlike other animals, are capable of self-healing. Marsupial biology is gaining attention as a result of these distinguishing characteristics.

The authors noted that the opossum—the first marsupial to have its entire genome sequenced—is used as a model species for basic marsupial biology research due to a number of experimentally advantageous traits.

To develop a fertilised egg with a genome that has been modified, the embryo must be transferred to a surrogate mother. The researchers successfully implanted a fertilised egg into the uterus of a fertile female opossum, as mice and rats do. This is the first time that marsupial embryo transfer has been demonstrated.

Typically, the fluid required for genome editing is injected into the fertilised egg using a fine needle. The injection needle, on the other hand, cannot penetrate the fertilised egg of the opossum because it is coated by a thick layer of proteins and a hard shell-like structure. “One of the keys to our success,” Hiroshi Kiyonari, PhD, team leader at RIKEN BDR’s laboratory for animal resources and genetic engineering, explained, “was the use of a piezoelectronic element in conjunction with the needle, which enabled the needle to penetrate the egg’s hard shell coat and thick layer surrounding it. Thus, the piezo has made it possible to implant zygotes without causing significant damage.”

After establishing the approach, researchers can concentrate on marsupial biology-related problems. “Marsupials are one of three extant mammalian subclasses, and they have a number of characteristics that set them apart from other mammals,” Kiyonari added. “Now that our proof-of-concept experiment has established the technology, future research can focus on developing genetically modified marsupials with implications for mammalian embryology, genomic imprinting, reproduction, neuroscience, immunogenetics, cancer biology, and even comparative evolution.”

Reference
Targeted gene disruption in a marsupial, Monodelphis domestica, by CRISPR/Cas9 genome editing. Current Biology. Kiyonari et al.DOI:https://doi.org/10.1016/j.cub.2021.06.056