Abstract

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.

Highlights

  • If a donor DNA containing longer genes (>1 kb) or single-stranded sequences (>200 bp) with homology to the target region is present, it will be introduced into the double-strand breaks (DSBs) site through homology-directed repair (HDR), a cellular mechanism that enables the precise recovery of the DSB

  • Complementary DNA expression constructs for the sex-determining region Y (Sry) protein, which is important for female-to-male sex-reversal, was injected into isolated genital ridge tissues, which were subjected to magnetically induced transfection

  • We have described the applicability of clustered regularly interspaced short palindromic repeats (CRISPR)/Caspase 9 (Cas9) system for producing GE

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Summary

Genome-Editing Technology

Genome-editing techniques involve the use of sequence-specific nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) that make it possible to induce modifications in a predefined region of the genome [1]. The embryos resulting from in vitro gene delivery into isolated zygotes/2-cell embryos or ICSI using GE sperm are transferred to the reproductive tracts of pseudo-pregnant female mice to enable their development to full-term. This procedure, invariably entails ex vivo handling of cells and embryos. In 2015, a novel in vivo approach called “genome-editing via oviductal nucleic acids delivery (GONAD)” was reported by Takahashi et al [16] This technique is performed by intraoviductal injection of a solution containing genome-editing reagents and subsequently by in vivo EP of the oviducts of a pregnant female at the 2-cell stage. Ex Vivo Delivery of Genome-Editing Components into Zygotes/2-Cell Embryos, PGCs, and Spermatogonial Stem Cells

Microinjection Technique
EP Technique
Gene Delivery to PGCs in the Genital Ridges
Gene Delivery to Spermatogonial Stem Cells
Infection of Preimplantation Embryos with AAVs
Gene Delivery to Postimplantation Embryos at Somite Stage
TPGD-GEF Technique
In Utero Gene Delivery
Conclusions
Methods
Findings
Full Text
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