Genome editing has been used to alter the genomes of species from plants to humans. Now, a group of researchers from the RIKEN Center for Biosystems Dynamics Research (BDR) have added a new animal to the list of animals that have been genetically engineered using CRISPR-Cas9, through the first successful demonstration of gene knockout in a marsupial.

This work is published in Current Biology, in the paper, “Targeted gene disruption in a marsupial, Monodelphis domestica, by CRISPR/Cas9 genome editing.

The researchers targeted a gene responsible for making body pigments; the animal lacks color when the gene is disrupted. More specifically, the team demonstrated “the generation of gene knockout opossums at the Tyr locus by microinjection of pronuclear stage zygotes using CRISPR/Cas9 genome editing, along with germline transmission of the edited alleles to the F1 generation.”

Among marsupials, which have multiple biologically unique characteristics, the gray short-tailed opossum (Monodelphis domestica) is one of few established laboratory models. The opossum develops without a functional placenta, and pups are born prematurely. Like humans, but not other non-marsupial mammals, the opossum gets skin cancer simply by exposure to ultraviolet light. Also unlike other mammals, newborn opossum pups with spinal cord injuries have the ability to naturally heal themselves. Because of these unique characteristics, studying marsupial biology is gaining interest.

Due to many experimentally advantageous features, the authors noted that the opossum—the first marsupial to have its entire genome sequenced—is used as a prototype species for basic research on marsupial biology.

Transplant of the embryo into a surrogate mother is required to generate a genome-edited fertilized egg. As is done in mice and rats, the researchers transferred the fertilized egg into the uterus of a fertile female opossum, and successfully obtained pups. This is the first case that embryo transfer technology has been established in marsupials.

Usually, the solution required for genome editing is injected into the fertilized egg using a fine needle. However, since the fertilized egg of the opossum is surrounded by a thick layer of proteins and a hard shell-like structure, the injection needle cannot penetrate it. “One of the tricks to our success,” Hiroshi Kiyonari, PhD, team leader in the laboratory for animal resources and genetic engineering at RIKEN BDR explained, “was using a piezoelectronic element along with the needle, which allowed the needle to penetrate the hard shell coat and thick layer surrounding the egg. The piezo has thus made it possible to inject zygotes without significant damage.”

Now that the process has been established, researchers can focus on answering questions regarding marsupial biology. “Marsupials represent one of three extant mammalian subclasses with a number of unique characteristics not shared by other mammals,” noted Kiyonari. “Having established the technology in this proof-of-concept experiment, future studies can create genetically modified marsupials that will impact the fields of mammalian embryology, genomic imprinting, reproduction, neurobiology, immunogenetics, cancer biology, and even comparative evolution.”

Graphical abstract for Kiyonari et al.