Genome-edited animals are widely used in various fields of research. Recent advances in the field of genome-editing, especially the CRISPR/Cas9 system (Clustered Regulatory Interspaced Short Palindromic Repeats /CRISPR associated protein), have enabled rapid generation of genome-edited animals with ease. However, the procedure for producing such animals involves multiple complicated steps and also necessitates special techniques, such as handling zygotes ex vivo. Many researchers desire a simple and convenient method for generating genome-edited animals.
The laboratory rat (Rattus norvegicus) is a common model of human diseases. Although there are several advantages compared with using mice, technology for handling the rat genome has lagged behind. A new, more simple method for genome editing in rats is now available, namely rGONAD (rat Genome-editing via Oviductal Nucleic Acids Delivery).
Although the GONAD method has so far been successfully implemented in mice and rats, this concept can be adapted for other animals.
The rGONAD method involves two key steps: an injection with the solution containing Cas9 protein, guide RNA and single strand DNA (ssDNA) into the oviduct, followed by electroporation. Rats that have undergone rGONAD are bred as usual until birth, where offspring would be genome-edited and used for further analyses. Moreover, rGONAD is highly efficient in both knock-out and knock-in systems.
The current standard method for genome-engineering in animals involves three major steps: isolation of zygotes from females, micromanipulation ex vivo and then transfer into pseudopregnant females. These steps are complicated and require skillful training to perform; rGONAD overcomes these caveats.
Furthermore, in the ethical context of the 3Rs (Reduction, Replacement, and Refinement), the standard method consumes many females, whereas female rats can be recycled in the rGONAD system. Although the GONAD method has so far been successfully implemented in mice and rats, this concept can be adapted for other animals. Therefore, GONAD is a powerful method for genome-editing in animal research.