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      Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System

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          Abstract

          In recent years, genome engineering technology has provided unprecedented opportunities for site-specific modification of biological genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 is one such means that can target a specific genome locus. It has been applied in human cells and many other organisms. Meanwhile, to efficiently enrich targeted cells, several surrogate systems have also been developed. However, very limited information exists on the application of CRISPR/Cas9 in chickens. In this study, we employed the CRISPR/Cas9 system to induce mutations in the peroxisome proliferator-activated receptor-γ ( PPAR-γ), ATP synthase epsilon subunit ( ATP5E), and ovalbumin ( OVA) genes in chicken DF-1 cells. The results of T7E1 assays showed that the mutation rate at the three different loci was 0.75%, 0.5%, and 3.0%, respectively. In order to improve the mutation efficiency, we used the Puro R gene for efficient enrichment of genetically modified cells with the surrogate reporter system. The mutation rate, as assessed via the T7E1 assay, increased to 60.7%, 61.3%, and 47.3%, and subsequent sequence analysis showed that the mutation efficiency increased to 94.7%, 95%, and 95%, respectively. In addition, there were no detectable off-target mutations in three potential off-target sites using the T7E1 assay. As noted above, the CRISPR/Cas9 system is a robust tool for chicken genome editing.

          Most cited references20

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          Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects.

          Bacterial RNA-directed Cas9 endonuclease is a versatile tool for site-specific genome modification in eukaryotes. Co-microinjection of mouse embryos with Cas9 mRNA and single guide RNAs induces on-target and off-target mutations that are transmissible to offspring. However, Cas9 nickase can be used to efficiently mutate genes without detectable damage at known off-target sites. This method is applicable for genome editing of any model organism and minimizes confounding problems of off-target mutations.
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            Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly.

            Broad applications of zinc finger nuclease (ZFN) technology-which allows targeted genome editing-in research, medicine, and biotechnology are hampered by the lack of a convenient, rapid, and publicly available method for the synthesis of functional ZFNs. Here we describe an efficient and easy-to-practice modular-assembly method using publicly available zinc fingers to make ZFNs that can modify the DNA sequences of predetermined genomic sites in human cells. We synthesized and tested hundreds of ZFNs to target dozens of different sites in the human CCR5 gene-a co-receptor required for HIV infection-and found that many of these nucleases induced site-specific mutations in the CCR5 sequence. Because human cells that harbor CCR5 null mutations are functional and normal, these ZFNs might be used for (1) knocking out CCR5 to produce T-cells that are resistant to HIV infection in AIDS patients or (2) inserting therapeutic genes at "safe sites" in gene therapy applications.
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              The DF-1 chicken fibroblast cell line: transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses.

              DF-1 is a continuous cell line of chicken embryo fibroblasts. The cells are free of endogenous sequences related to avian sarcoma and leukosis viruses and have normal fibroblastic morphology. DF-1 cells support the replication of avian retroviruses; diverse oncogenes induce foci of oncogenic transformation on monolayers of DF-1, and avian leukosis viruses of envelope subgroups B, D, and C induce cell death and form plaques. The new cell line will greatly facilitate studies on oncogenic transformation and cell killing by avian viruses. Copyright 1998 Academic Press.
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                Author and article information

                Journal
                G3 (Bethesda)
                Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes|Genomes|Genetics
                Genetics Society of America
                2160-1836
                10 February 2016
                April 2016
                : 6
                : 4
                : 917-923
                Affiliations
                [1]College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China
                Author notes
                [1 ]Corresponding authors: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China. E-mails: Zhangzhy@ 123456nwsuaf.edu.cn and 553527211@ 123456126.com
                Article
                GGG_027706
                10.1534/g3.116.027706
                4825661
                26869617
                cfe0ea41-6bc4-4d58-834c-cf901dd9cc96
                Copyright © 2016 Bai et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 02 December 2015
                : 01 February 2016
                Page count
                Pages: 7
                Categories
                Genomic Selection

                Genetics
                crispr/cas9 system,surrogate reporter,chicken df-1 cell,mutant efficiency,off-target,genpred,shared data resource,genomic selection

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