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      A Golgi-derived vesicle potentiates PtdIns4P to PtdIns3P conversion for endosome fission

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          Genome engineering using the CRISPR-Cas9 system.

          Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.
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            Mechanisms of clathrin-mediated endocytosis

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              Mechanisms of endocytosis.

              Endocytic mechanisms control the lipid and protein composition of the plasma membrane, thereby regulating how cells interact with their environments. Here, we review what is known about mammalian endocytic mechanisms, with focus on the cellular proteins that control these events. We discuss the well-studied clathrin-mediated endocytic mechanisms and dissect endocytic pathways that proceed independently of clathrin. These clathrin-independent pathways include the CLIC/GEEC endocytic pathway, arf6-dependent endocytosis, flotillin-dependent endocytosis, macropinocytosis, circular doral ruffles, phagocytosis, and trans-endocytosis. We also critically review the role of caveolae and caveolin1 in endocytosis. We highlight the roles of lipids, membrane curvature-modulating proteins, small G proteins, actin, and dynamin in endocytic pathways. We discuss the functional relevance of distinct endocytic pathways and emphasize the importance of studying these pathways to understand human disease processes.
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                Author and article information

                Contributors
                Journal
                Nature Cell Biology
                Nat Cell Biol
                Springer Science and Business Media LLC
                1465-7392
                1476-4679
                June 28 2021
                Article
                10.1038/s41556-021-00704-y
                34183801
                d9b7c38b-96ee-4943-bc7b-8683fc48e623
                © 2021

                https://www.springer.com/tdm

                https://www.springer.com/tdm

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