Recent Advances in In Vivo Genome Editing Targeting Mammalian Preimplantation Embryos

Abstract

CRISPR-based genome engineering has been widely used for producing gene-modified animals such as mice and rats, to explore the function of a gene of interest and to create disease models. However, it always requires the ex vivo handling of preimplantation embryos, as exemplified by the microinjection of genome editing components into zygotes or in vitro electroporation of zygotes in the presence of genome editing components, and subsequent cultivation of the treated embryos prior to egg transfer to the recipient females. To avoid this ex vivo process, we have developed a novel method called genome-editing via oviductal nucleic acids delivery (GONAD) or improved GONAD (i-GONAD), which enables in situ genome editing of zygotes present in the oviductal lumen of a pregnant female. This technology does not require any ex vivo handling of preimplantation embryos or preparation of recipient females and vasectomized males, all of which are often laborious and time-consuming. In this chapter, recent advances in the development of GONAD/i-GONAD will be described.

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

“…Since the reports on GONAD/i-GONAD in 2015 and 2018, several improvements have been made in these systems, including i-GONAD, applied at the early zygote stage ("ezi-GONAD") [45], two-step i-GONAD (si-GONAD) [20], and i-GONAD allowing tagging at a desired endogenous gene (tagi-GONAD) [32,44]. These technologies now create pups with KO or KI phenotypes [12]. Thus, GONAD/i-GONAD and their derivatives are now recognized as possible alternatives to pre-existing systems based on the ex vivo handling of zygotes because they have many advantages over the previous methods because of the reduced number of females used (therefore fitting the animal welfare 3R principle) and the absence of laborious tasks required for ex vivo handling of embryos (such as embryo isolation, MI or in vitro EP procedure, cultivation of sources, and ET to pseudopregnant recipient females).…”

“…Since the report of GONAD/i-GONAD, many researchers have reproduced this technology using mice, rats, and hamsters, and some modifications have been made to these technologies [12]. For examples, these include production of KO or KI mice using ssODN as a donor [6,7,, large deletion (LD) of a target site in chromosomes [7,22,37], maintenance of lethal mutant mice using an inversion balancer identified from the C3H/HeJJcl strain [38], production of rodents with floxed alleles [20,22,31] or long DNA fragments at a target locus [7], epitope-tagging at a target gene [32] or in situ somatic gene replacement in oviductal epithelium [39].…”

“…More than 30 genes have been successfully modified by GONAD/i-GONAD [12]. GONAD/i-GONAD also enables the simultaneous transfection of epithelial cells lining the oviductal lumen [7,8,17].…”

“…GONAD/i-GONAD also enables the simultaneous transfection of epithelial cells lining the oviductal lumen [7,8,17]. Since recent advance in GONAD/i-GONAD-based production of genome-edited animals was already summarized in our previous review paper [12], we will show the recent i-GONAD-related works (reported on 2022 to 2023) in Table 1 and also focus on the following subjects: 1) present and future modification of GONAD/i-GONAD, 2) AAV-based GONAD, 3) possible application of GONAD/i-GONAD to manipulate the genome of animals other than mice, 4) the utility of these technologies to examine female reproductive systems, such as oviductal function related to the shape formation (coil or non coil) of the oviduct, embryo transfer and embryo survival, and 5) the utility of these technologies to generate ovarian tumors and exploration of the mechanism underlying carcinogenesis. Pairs of guide RNAs were designed to generate controlled deletions that led to the absence of protein; in vitro EP or i-GONAD resulted in more than 90% and 30% genome-edited newborn animals, respectively, for each locus.…”