Abstract
One of the most powerful ways to test the function of a protein is to characterize the consequences of its deletion. In the past, this has involved inactivation of the gene by homologous recombination either in the germline or later through conditional deletion. RNA interference (RNAi) provides an alternative way to knock down proteins, but both of these approaches have their limitations. Recently, the CRISPR/Cas9 system has suggested another way to selectively inactivate genes. We have now tested this system in postmitotic neurons by targeting two well-characterized synaptic proteins, the obligatory GluN1 subunit of the NMDA receptor and the GluA2 subunit of the AMPA receptor. Expression of CRISPR/Cas9 in hippocampal slice cultures completely eliminated NMDA receptor and GluA2 function. CRISPR/Cas9 thus provides a powerful tool to study the function of synaptic proteins.
Copyright © 2014 Elsevier Inc. All rights reserved.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Animals
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Animals, Newborn
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Cells, Cultured
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Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
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Excitatory Amino Acid Agents / pharmacology
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Excitatory Postsynaptic Potentials / drug effects
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Excitatory Postsynaptic Potentials / genetics
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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Hippocampus / cytology
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Humans
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In Vitro Techniques
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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N-Methylaspartate / pharmacology
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Nerve Tissue Proteins / genetics
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Nerve Tissue Proteins / metabolism*
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Neurons / metabolism*
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Quinoxalines / pharmacology
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RNA / genetics
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RNA / metabolism
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Rats
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Receptors, AMPA / genetics
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Receptors, AMPA / metabolism*
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Receptors, N-Methyl-D-Aspartate / genetics
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Receptors, N-Methyl-D-Aspartate / metabolism*
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Red Fluorescent Protein
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Synapses / genetics
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Synapses / metabolism*
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Synaptophysin / metabolism
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alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / pharmacology
Substances
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Excitatory Amino Acid Agents
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GRIN1 protein, human
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Luminescent Proteins
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Nerve Tissue Proteins
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Quinoxalines
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Receptors, AMPA
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Receptors, N-Methyl-D-Aspartate
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Synaptophysin
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2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline
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Green Fluorescent Proteins
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RNA
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N-Methylaspartate
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alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid
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glutamate receptor ionotropic, AMPA 2