Recent Genome-Editing Approaches toward Post-Implanted Fetuses in Mice

Abstract

Genome editing, as exemplified by the CRISPR/Cas9 system, has recently been employed to effectively generate genetically modified animals and cells for the purpose of gene function analysis and disease model creation. There are at least four ways to induce genome editing in individuals: the first is to perform genome editing at the early preimplantation stage, such as fertilized eggs (zygotes), for the creation of whole genetically modified animals; the second is at post-implanted stages, as exemplified by the mid-gestational stages (E9 to E15), for targeting specific cell populations through in utero injection of viral vectors carrying genome-editing components or that of nonviral vectors carrying genome-editing components and subsequent in utero electroporation; the third is at the mid-gestational stages, as exemplified by tail-vein injection of genome-editing components into the pregnant females through which the genome-editing components can be transmitted to fetal cells via a placenta-blood barrier; and the last is at the newborn or adult stage, as exemplified by facial or tail-vein injection of genome-editing components. Here, we focus on the second and third approaches and will review the latest techniques for various methods concerning gene editing in developing fetuses.

Keywords: CRISPR/Cas9; electroporation; fetuses; genome editing; in utero gene delivery; indels; knock-in; knock-out; tail-vein injection; transplacental gene delivery.

Figure 1

Gene delivery into early-to-mid-gestational fetuses. (A) Gene delivery into somite-stage embryos. Introduction of exogenous nucleic acids into the abdomen of anesthetized pregnant female is possible through a glass micropipette under a dissecting microscope after exposure of the uterus. (B) Gene delivery into fetuses at embryonic day (E) 12.5. At E12.5, the fetus (embryo) is visible through the yolk sac (YS) upon surgical dissection of the uterus under a dissecting microscope. Thus, it is possible to administer intrabrain (a), intraamniotic (b), intraplacental (c), intrahepatic (d), intracardiac (e), intravitelline (f), and skin (g) injections of genome-editing components using a micropipette for in utero gene delivery. (C) Gene delivery into fetuses using transplacental gene delivery (TPGD). Tail-vein injection of a solution containing nucleic acids into pregnant female mice is also a useful in vivo approach to introduce nucleic acids into E9.5–12.5 fetuses. This figure was drawn in-house and reproduced with permission from Sato et al. [6], published by MDPI, 2020.

Figure 2

Substances introduced via intraamniotic injection can be taken up by a fetus (at E16). According to Ricciardi et al. [45], intraamniotic injection at E15 did not lead to any detectable accumulation of materials injected within the fetus. However, injection at E16—the expected time of onset of pronounced fetal breathing and swallowing—resulted in material accumulation in the fetal lung and gut. This figure was drawn in-house and reproduced based on Alapati et al. [46].

Figure 3

Transplacental gene delivery (TPGD) at E12.5. (A) Localization of trypan blue in the isolated fetus with placenta and yolk sac (YS) one day after tail-vein injection into a pregnant female at E12.5. Notably, almost trypan blue was trapped in the placenta and YS (arrows). The photo is based on the picture from Kikuchi et al. [64] published by Nature Publishing group, 2002. (B) Hypothetical mechanism of TPGD as suggested by Kikuchi et al. [64]. Following TPGD on E12.5, when placental circulation is established, intravenously injected plasmid DNA/lipid complexes may be transferred from maternal blood to the fetus via at least two routes. Flow via the placenta to the embryo is indicated by the arrows in area A; injected plasmid DNA is transferred beyond the blood–placenta barrier and enters the umbilical cord. Flow from the decidua to the YS is indicated by the arrows in area B; some DNA becomes trapped in YS and is transferred to the embryo after the establishment of functional placental circulation. This figure was drawn in-house and reproduced with permission from Nakamura et al. [8] published by MDPI, 2019. (C) Schematic representation of the experimental outline of TPGD-GEF. At E12.5, a solution containing plasmid DNA complexed with gene delivery reagent (i.e., FuGENE6) was intravenously administered to the pregnant female mice. Two days after the in vivo transfection, fetuses were dissected to check the expression of the introduced DNA. (D) Decreased expression of EGFP-derived fluorescence in the TPGD-GEF-treated fetus. In the intact control fetus (Control group), the heart exhibited strong fluorescence, whereas some TPGD-GEF-treated fetuses exhibited reduced fluorescence in their hearts (Experimental group). The number of pixels of “luminance” in each area is analyzed in the software’s histogram function (Adobe Photoshop Elements 2018) and plotted as a graph (right panel). The areas analyzed are the three sites (head, heart, and base of the tail) shown in the boxes of the figure. Notably, fluorescence in the genome-edited fetal heart area was greatly reduced compared to that in unedited fetuses, suggesting extensive genome editing in the hearts of genome-edited fetuses. The figures in (C,D) were drawn in-house and reproduced with permission from Nakamura et al. [7], published by Wiley, 2019.