title: Nucleic acids delivery methods for genome editing in zygotes and embryos: the old, the new, and the old-new.
abstract: In the recent years, sequence-specific nucleases such as ZFNs, TALENs, and CRISPR/Cas9 have revolutionzed the fields of animal genome editing and transgenesis. However, these new techniques require microinjection to deliver nucleic acids into embryos to generate gene-modified animals. Microinjection is a delicate procedure that requires sophisticated equipment and highly trained and experienced technicians. Though over a dozen alternate approaches for nucleic acid delivery into embryos were attempted during the pre-CRISPR era, none of them became routinely used as microinjection. The addition of CRISPR/Cas9 to the genome editing toolbox has propelled the search for novel delivery approaches that can obviate the need for microinjection. Indeed, some groups have recently developed electroporation-based methods that have the potential to radically change animal transgenesis. This review provides an overview of the old and new delivery methods, and discusses various strategies that were attempted during the last three decades. In addition, several of the methods are re-evaluated with respect to their suitability to deliver genome editing components, particularly CRISPR/Cas9, to embryos.
authors.affiliation: Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Kagoshima, 890-8544, Japan. masasato@ms.kagoshima-u.ac.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sato
authors.affiliation: Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa, 259 1193, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Ohtsuka
authors.affiliation: Animal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Ibaraki, 305-8602, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Developmental Neuroscience, Munroe-Meyer Institute, University of Nebraska Medical Center, Omaha, NE, 68198, USA. cgurumurthy@unmc.edu.|Mouse Genome Engineering Core Facility, University of Nebraska Medical Center, Omaha, NE, 68198, USA. cgurumurthy@unmc.edu.
authors.forename: Channabasavaiah B
authors.initials: CB
authors.lastname: Gurumurthy
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D005260:Female; D005818:Genetic Engineering; D006801:Humans; D009696:Nucleic Acids / Q000235:genetics; D011247:Pregnancy; D017393:RNA Editing / Q000235:genetics; D015053:Zygote
chemical_list: D009696:Nucleic Acids
keywords: CRISPR/Cas; Genome editing; Mcroinjection; Nucleic acids delivery; Pronuclaer injection; Transgenic