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      Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing 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 transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified 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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          Most cited references 42

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          NIH Image to ImageJ: 25 years of image analysis.

          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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            2012. NIH Image to ImageJ: 25 years of image analysis

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              CRISPR RNA-guided activation of endogenous human genes

              Catalytically inactive CRISPR-associated 9 nuclease (dCas9) can be directed by short guide RNAs (gRNAs) to repress endogenous genes in bacteria and human cells. Here we show that a dCas9-VP64 transcriptional activation domain fusion protein can be directed by single or multiple gRNAs to increase expression of specific endogenous human genes. These results provide an important proof-of-principle that CRISPR-Cas systems can be used to target heterologous effector domains in human cells.
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                Author and article information

                Journal
                Nat. Biotechnol.
                Nature biotechnology
                Springer Nature
                1546-1696
                1087-0156
                Jan 2015
                : 33
                : 1
                Affiliations
                [1 ] 1] Department of Chemistry &Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
                [2 ] 1] Department of Otology and Laryngology, Harvard Medical School, Boston, Massachusetts, USA. [2] Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA. [3] Department of Otology and Skull Base Surgery, Eye, Ear, Nose and Throat Hospital, Shanghai Medical College, Fudan University, Shanghai, China. [4] Key Laboratory of Health Ministry for Hearing Medicine, Shanghai, China.
                [3 ] 1] Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [2] Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [3] Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [4] Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.
                [4 ] 1] Department of Otology and Laryngology, Harvard Medical School, Boston, Massachusetts, USA. [2] Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA.
                Article
                nbt.3081 NIHMS637767
                10.1038/nbt.3081
                4289409
                25357182

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