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

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      Abstract

      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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      For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the analysis of scientific images. We discuss the origins, challenges and solutions of these two programs, and how their history can serve to advise and inform other software projects.
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        RNA-guided human genome engineering via Cas9.

        Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
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          One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.

          Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty--8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions. Copyright © 2013 Elsevier Inc. All rights reserved.
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            Author and article information

            Affiliations
            [1 ]Department of Chemistry & Chemical Biology, Harvard University, Cambridge, Massachusetts, USA
            [2 ]Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA
            [3 ]Department of Otolaryngology, Eaton Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA
            [4 ]Program in Neuroscience, Harvard Medical School, Boston, Massachusetts, USA
            [5 ]Department of Otology and Skull Base Surgery, Eye, Ear, Nose and Throat Hospital, Shanghai Medical College, Fudan University, Shanghai, China
            [6 ]Key Laboratory of Health Ministry for Hearing Medicine, Shanghai, China
            [7 ]Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA
            [8 ]Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA
            [9 ]Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
            [10 ]Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA
            Author notes
            [* ]Correspondence should be addressed to D.R.L. ( drliu@ 123456fas.harvard.edu )
            Journal
            9604648
            20305
            Nat Biotechnol
            Nat. Biotechnol.
            Nature biotechnology
            1087-0156
            1546-1696
            24 October 2014
            30 October 2014
            January 2015
            01 July 2015
            : 33
            : 1
            : 73-80
            25357182
            4289409
            10.1038/nbt.3081
            NIHMS637767
            Categories
            Article

            Biotechnology

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