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      CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation

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          Abstract

          Virus-free gene editing possible at chromosomes through stretching cells in a micropost array, even for hard-to-transfect cells.

          Abstract

          The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) nuclease system represents an efficient tool for genome editing and gene function analysis. It consists of two components: single-guide RNA (sgRNA) and the enzyme Cas9. Typical sgRNA and Cas9 intracellular delivery techniques are limited by their reliance on cell type and exogenous materials as well as their toxic effects on cells (for example, electroporation). We introduce and optimize a microfluidic membrane deformation method to deliver sgRNA and Cas9 into different cell types and achieve successful genome editing. This approach uses rapid cell mechanical deformation to generate transient membrane holes to enable delivery of biomaterials in the medium. We achieved high delivery efficiency of different macromolecules into different cell types, including hard-to-transfect lymphoma cells and embryonic stem cells, while maintaining high cell viability. With the advantages of broad applicability across different cell types, particularly hard-to-transfect cells, and flexibility of application, this method could potentially enable new avenues of biomedical research and gene targeting therapy such as mutation correction of disease genes through combination of the CRISPR-Cas9–mediated knockin system.

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          Most cited references 19

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          Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.

          Single murine and human intestinal stem cells can be expanded in culture over long time periods as genetically and phenotypically stable epithelial organoids. Increased cAMP levels induce rapid swelling of such organoids by opening the cystic fibrosis transmembrane conductor receptor (CFTR). This response is lost in organoids derived from cystic fibrosis (CF) patients. Here we use the CRISPR/Cas9 genome editing system to correct the CFTR locus by homologous recombination in cultured intestinal stem cells of CF patients. The corrected allele is expressed and fully functional as measured in clonally expanded organoids. This study provides proof of concept for gene correction by homologous recombination in primary adult stem cells derived from patients with a single-gene hereditary defect. Copyright © 2013 Elsevier Inc. All rights reserved.
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            Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype.

             Hao Yin,  Wen Xue,  Sidi Chen (2014)
            We demonstrate CRISPR-Cas9-mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. Delivery of components of the CRISPR-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in ∼1/250 liver cells. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype. Our study indicates that CRISPR-Cas9-mediated genome editing is possible in adult animals and has potential for correction of human genetic diseases.
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              CRISPR-mediated direct mutation of cancer genes in the mouse liver

              The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem (ES) cells 1 . Here we describe a new method of cancer model generation using the CRISPR/Cas system in vivo in wild-type mice. We have used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) 2–4 to the liver and directly target the tumor suppressor genes Pten 5 and p53 6 , alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology 7, 8 . Simultaneous targeting of Pten and p53 induced liver tumors that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumor tissue revealed insertion or deletion (indel) mutations of the tumor suppressor genes, including bi-allelic mutations of both Pten and p53 in tumors. Furthermore, co-injection of Cas9 plasmids harboring sgRNAs targeting the β-Catenin gene (Ctnnb1) and a single-stranded DNA (ssDNA) oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-Catenin. This study demonstrates the feasibility of direct mutation of tumor suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics.
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                Author and article information

                Journal
                Sci Adv
                Sci Adv
                SciAdv
                advances
                Science Advances
                American Association for the Advancement of Science
                2375-2548
                August 2015
                14 August 2015
                : 1
                : 7
                Affiliations
                [1 ]Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030, USA.
                [2 ]Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY 10065, USA.
                [3 ]Department of Pathology and Genomic Medicine and Cancer Pathology Laboratory, Houston Methodist Hospital and Houston Methodist Research Institute, Houston, TX 77030, USA.
                [4 ]Department of Experimental Radiation Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
                [5 ]Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
                Author notes
                [* ]Corresponding author. E-mail: lqin@ 123456houstonmethodist.org
                Article
                1500454
                10.1126/sciadv.1500454
                4643799
                Copyright © 2015, The Authors

                This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

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                Luningning Borromeo

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