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      Generation of CRISPR/Cas9-mediated lactoferrin-targeted mice by pronuclear injection of plasmid pX330

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          Abstract

          Lactoferrin is a member of the transferrin family of multifunctional iron binding glycoproteins. While numerous physiological functions have been described for lactoferrin, the mechanisms underlying these functions are not clear. To further study the functions and mechanisms of lactoferrin, we modified the lactoferrin promoter of mice using the CRISPR/Cas9 system to reduce or eliminate lactoferrin expression. Seven mice with lactoferrin promoter mutations were obtained with an efficiency of 24% (7/29) by injecting the plasmid pX330, expressing a small guide RNA and human codon-optimized SpCas9, into fertilized eggs of mice. Plasmid integration and off-targeting of pX330 were not detected. These results confirmed that pronuclear injection of a circular plasmid is a feasible and efficient method for targeted mutagenesis in mice.

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          Most cited references 16

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          Efficient In Vivo Genome Editing Using RNA-Guided Nucleases

          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have evolved in bacteria and archaea as a defense mechanism to silence foreign nucleic acids of viruses and plasmids. Recent work has shown that bacterial type II CRISPR systems can be adapted to create guide RNAs (gRNAs) capable of directing site-specific DNA cleavage by the Cas9 nuclease in vitro. Here we show that this system can function in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies comparable to those obtained using ZFNs and TALENs for the same genes. RNA-guided nucleases robustly enabled genome editing at 9 of 11 different sites tested, including two for which TALENs previously failed to induce alterations. These results demonstrate that programmable CRISPR/Cas systems provide a simple, rapid, and highly scalable method for altering genes in vivo, opening the door to using RNA-guided nucleases for genome editing in a wide range of organisms.
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            Generation of gene-modified mice via Cas9/RNA-mediated gene targeting.

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              Lactoferrin--a multifunctional protein with antimicrobial properties.

              Lactoferrin is a member of the transferrin family of iron-binding proteins. Numerous functions have been reported and continue to be reported for the protein, some of which are related to its iron-binding properties. Its extensive antimicrobial activities were originally attributed to its ability to sequester essential iron, however, it is now established that it possesses bactericidal activities as a result of a direct interaction between the protein or lactoferrin-derived peptides. This article reviews the antimicrobial activities of lactoferrin and discusses the potential mode of action of lactoferrin-derived cationic peptides against Gram-negative bacteria in the light of recent studies.
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                Author and article information

                Journal
                Front. Agr. Sci. Eng.
                FASE
                CN10-1204/S
                Frontiers of Agricultural Science and Engineering
                Higher Education Press (4 Huixin Dongjie, Chaoyang District, Beijing 100029, China )
                2095-7505
                2015
                : 2
                : 3
                : 242-248
                Affiliations
                1. State Key Laboratory of Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing 100193, China
                2. College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China
                Author notes
                ninglcau@cau.edu.cn
                qymeng@cau.edu.cn
                Article
                10.15302/J-FASE-2015059

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Categories
                RESEARCH ARTICLE

                plasmid pX330, promoter, lactoferrin, CRISPR/Cas9

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