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Sequential i-GONAD: An Improved In Vivo Technique for CRISPR/Cas9-Based Genetic Manipulations in Mice
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Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice

1
Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima 890-8544, Japan
2
Laboratory Animal Facilities & Services, Preeminent Medical Photonics Education & Research Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan
3
Division of Biomedical Engineering, National Defense Medical College Research Institute, Saitama 359-8513, Japan
*
Author to whom correspondence should be addressed.
Cells 2020, 9(4), 799; https://doi.org/10.3390/cells9040799
Received: 28 February 2020 / Revised: 21 March 2020 / Accepted: 23 March 2020 / Published: 26 March 2020
(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application)
The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) systems that occur in nature as microbial adaptive immune systems are considered an important tool in assessing the function of genes of interest in various biological systems. Thus, development of efficient and simple methods to produce genome-edited (GE) animals would accelerate research in this field. The CRISPR/Cas9 system was initially employed in early embryos, utilizing classical gene delivery methods such as microinjection or electroporation, which required ex vivo handling of zygotes before transfer to recipients. Recently, novel in vivo methods such as genome editing via oviductal nucleic acid delivery (GONAD), improved GONAD (i-GONAD), or transplacental gene delivery for acquiring genome-edited fetuses (TPGD-GEF), which facilitate easy embryo manipulation, have been established. Studies utilizing these techniques employed pregnant female mice for direct introduction of the genome-editing components into the oviduct or were dependent on delivery via tail-vein injection. In mice, embryogenesis occurs within the oviducts and the uterus, which often hampers the genetic manipulation of embryos, especially those at early postimplantation stages (days 6 to 8), owing to a thick surrounding layer of tissue called decidua. In this review, we have surveyed the recent achievements in the production of GE mice and have outlined the advantages and disadvantages of the process. We have also referred to the past achievements in gene delivery to early postimplantation stage embryos and germ cells such as primordial germ cells and spermatogonial stem cells, which will benefit relevant research. View Full-Text
Keywords: genome editing; CRISPR/Cas9; zygotes; postimplantation embryos; fetuses; primordial germ cells; spermatogonial stem cells; GONAD; TPGD-GEF; adeno-associated virus genome editing; CRISPR/Cas9; zygotes; postimplantation embryos; fetuses; primordial germ cells; spermatogonial stem cells; GONAD; TPGD-GEF; adeno-associated virus
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Sato, M.; Takabayashi, S.; Akasaka, E.; Nakamura, S. Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice. Cells 2020, 9, 799.

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