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      Inducible Genome Editing with Conditional CRISPR/Cas9 Mice

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          Abstract

          Genetically engineered mouse models (GEMMs) are powerful tools by which to probe gene function in vivo, obtain insight into disease etiology, and identify modifiers of drug response. Increased sophistication of GEMMs has led to the design of tissue-specific and inducible models in which genes of interest are expressed or ablated in defined tissues or cellular subtypes. Here we describe the generation of a transgenic mouse harboring a doxycycline-regulated Cas9 allele for inducible genome engineering. This model provides a flexible platform for genome engineering since editing is achieved by exogenous delivery of sgRNAs and should allow for the modeling of a range of biological and pathological processes.

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          Most cited references 20

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          Efficient Delivery of Genome-Editing Proteins In Vitro and In Vivo

          Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape, and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains, or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcriptional activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.
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            Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells.

            Transgenic and gene-targeted mutant mice provide powerful tools for analysis of the cellular processes involved in early development and in the pathogenesis of many diseases. Here we describe a transgene integration strategy mediated by site-specific recombination that allows establishment of multiple embryonic stem (ES) cell lines carrying tetracycline-inducible genes targeted to a specific locus to assure predictable temporal and spatial expression in ES cells and mice. Using homologous recombination we inserted an frt homing site into which tetracycline-inducible transgenes can be integrated efficiently in the presence of FLPe recombinase. This strategy and the vectors described here are generally applicable to any locus in ES cells and should allow for the rapid production of mice with transgenes efficiently targeted to a defined site. (c) 2006 Wiley-Liss, Inc.
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              Expanding the Biologist's Toolkit with CRISPR-Cas9.

              Few discoveries transform a discipline overnight, but biologists today can manipulate cells in ways never possible before, thanks to a peculiar form of prokaryotic adaptive immunity mediated by clustered regularly interspaced short palindromic repeats (CRISPR). From elegant studies that deciphered how these immune systems function in bacteria, researchers quickly uncovered the technological potential of Cas9, an RNA-guided DNA cleaving enzyme, for genome engineering. Here we highlight the recent explosion in visionary applications of CRISPR-Cas9 that promises to usher in a new era of biological understanding and control.
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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
                08 March 2018
                May 2018
                : 8
                : 5
                : 1627-1635
                Affiliations
                [* ]Department of Biochemistry, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec
                []The Rosalind and Morris Goodman Cancer Research Center, McGill University, Montreal, Québec, Canada, H3G 1Y6
                [§ ]Department of Oncology, McGill University, Montreal, Québec, Canada, H3G 1Y6
                []Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec
                Author notes
                [1 ]To whom correspondence should be addressed: Department of Biochemistry, McGill University, Montreal, Québec, Canada, H3G 1Y6. E-mail: jerry.pelletier@ 123456mcgill.ca
                Article
                GGG_300327
                10.1534/g3.117.300327
                5940154
                29519936
                Copyright © 2018 Katigbak 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.

                Page count
                Figures: 5, Tables: 1, Equations: 0, References: 28, Pages: 9
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                Genetics

                conditional cas9 mouse, cas9 knock-in, mouse model, genome editing, crispr/cas9

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