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      Single-Cell Labeling Strategies to Dissect Neuronal Structures and Local Functions

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      Biology
      MDPI AG

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          Abstract

          The brain network consists of ten billion neurons and is the most complex structure in the universe. Understanding the structure of complex brain networks and neuronal functions is one of the main goals of modern neuroscience. Since the seminal invention of Golgi staining, single-cell labeling methods have been among the most potent approaches for dissecting neuronal structures and neural circuits. Furthermore, the development of sparse single-cell transgenic methods has enabled single-cell gene knockout studies to examine the local functions of various genes in neural circuits and synapses. Here, we review non-transgenic single-cell labeling methods and recent advances in transgenic strategies for sparse single neuronal labeling. These methods and strategies will fundamentally contribute to the understanding of brain structure and function.

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          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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            Genome editing. The new frontier of genome engineering with CRISPR-Cas9.

            The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics. Copyright © 2014, American Association for the Advancement of Science.
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              The green fluorescent protein.

              R Tsien (1998)
              In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. High-resolution crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiological indicators, biosensors, and photochemical memories.
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                Author and article information

                Contributors
                (View ORCID Profile)
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                Journal
                BBSIBX
                Biology
                Biology
                MDPI AG
                2079-7737
                February 2023
                February 16 2023
                : 12
                : 2
                : 321
                Article
                10.3390/biology12020321
                9953318
                36829594
                934fbe46-7084-4f95-823c-b0ea4fa35114
                © 2023

                https://creativecommons.org/licenses/by/4.0/

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