title: Efficient, complete deletion of synaptic proteins using CRISPR.
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.
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Incontro
authors.affiliation: Department of Physiology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Neurology, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Cedric S
authors.initials: CS
authors.lastname: Asensio
authors.affiliation: Department of Physiology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Neurology, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Edwards
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Physiology, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: roger.nicoll@ucsf.edu.
authors.forename: Roger A
authors.initials: RA
authors.lastname: Nicoll
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D018683:Excitatory Amino Acid Agents / Q000494:pharmacology; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016202:N-Methylaspartate / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D011810:Quinoxalines / Q000494:pharmacology; D012313:RNA / Q000235:genetics / Q000378:metabolism; D051381:Rats; D018091:Receptors, AMPA / Q000235:genetics / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000378:metabolism*; D013569:Synapses / Q000235:genetics / Q000378:metabolism*; D016708:Synaptophysin / Q000378:metabolism; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology; D000097573:Red Fluorescent Protein
chemical_list: D018683:Excitatory Amino Acid Agents; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D016202:N-Methylaspartate; D009419:Nerve Tissue Proteins; D011810:Quinoxalines; D012313:RNA; D018091:Receptors, AMPA; D016194:Receptors, N-Methyl-D-Aspartate; D016708:Synaptophysin; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D000097573:Red Fluorescent Protein; C485668:GRIN1 protein, human; C062865:2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline; C104722:glutamate receptor ionotropic, AMPA 2