Recent Advances in the Production of Genome-Edited Animals Using <i>i</i>-GONAD, a Novel <i>in vivo</i> Genome Editing System, and Its Possible Use for the Study of Female Reproductive Systems

Abstract

Gene-engineered animals created using gene-targeting technology have long been recognized as beneficial, valid, and valuable tools for exploring the function of a gene of interest, at least in early 2013. This approach, however, suffers from laborious and time-consuming tasks, such as the production of successfully targeted embryonic stem (ES) cells, their characterization, production of chimeric blastocysts carrying these gene-modified ES cells, and transplantation of those manipulated blastocysts to the recipient (pseudopregnant) females to deliver chimeric mice. Since the appearance of genome editing technology, which is now exemplified by the CRISPR/<em>Cas9</em> system, in late 2013, significant advances have been made in the generation of genome-edited animals through pronuclear microinjection (MI) of genome-editing components into fertilized eggs (zygotes) or electroporation (EP) of zygotes in the presence of these reagents. However, these procedures require the transfer of genome-edited embryos into the reproductive tracts of recipient females for further development. <u>G</u>enome editing via <u>o</u>viductal <u>n</u>ucleic <u>a</u>cids <u>d</u>elivery (GONAD) and its modified version, called “improved GONAD (<em>i</em>-GONAD),” were developed as an alternative to the MI- or EP-based genome-edited animal production and now recognized to be very convenient and straightforward as genome editing can only be performed <em>in</em> <em>vivo</em> (within the oviductal lumen where fertilized embryos exist). This system also enables the simultaneous transfection of epithelial cells <em>lining the oviductal lumen</em>. In this review, we summarize the recent advances in GONAD/<em>i</em>-GONAD and their derivatives and discuss the potential of these technologies to study various biological systems related to female reproduction.