title: Fluorescent protein tagging of endogenous protein in brain neurons using CRISPR/Cas9-mediated knock-in and in utero electroporation techniques.
abstract: Genome editing is a powerful technique for studying gene functions. CRISPR/Cas9-mediated gene knock-in has recently been applied to various cells and organisms. Here, we successfully knocked in an EGFP coding sequence at the site immediately after the first ATG codon of the β-actin gene in neurons in the brain by the combined use of the CRISPR/Cas9 system and in utero electroporation technique, resulting in the expression of the EGFP-tagged β-actin protein in cortical layer 2/3 pyramidal neurons. We detected EGFP fluorescence signals in the soma and neurites of EGFP knock-in neurons. These signals were particularly abundant in the head of dendritic spines, corresponding to the localization of the endogenous β-actin protein. EGFP knock-in neurons showed no detectable changes in spine density and basic electrophysiological properties. In contrast, exogenously overexpressed EGFP-β-actin showed increased spine density and EPSC frequency, and changed resting membrane potential. Thus, our technique provides a potential tool to elucidate the localization of various endogenous proteins in neurons by epitope tagging without altering neuronal and synaptic functions. This technique can be also useful for introducing a specific mutation into genes to study the function of proteins and genomic elements in brain neurons.
authors: Department of Molecular and Cellular Physiology, Institute of Medicine, Academic Assembly, Shinshu University, Nagano 390-8621, Japan.|Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Nagano 390-8621, Japan.|CREST, JST, Saitama 332-0012, Japan.
authors: Takeshi
authors: T
authors: Uemura
authors: Department of Molecular and Cellular Physiology, Institute of Medicine, Academic Assembly, Shinshu University, Nagano 390-8621, Japan.
authors: Takuma
authors: T
authors: Mori
authors: Department of Molecular and Cellular Physiology, Institute of Medicine, Academic Assembly, Shinshu University, Nagano 390-8621, Japan.
authors: Taiga
authors: T
authors: Kurihara
authors: Department of Molecular and Cellular Physiology, Institute of Medicine, Academic Assembly, Shinshu University, Nagano 390-8621, Japan.|CREST, JST, Saitama 332-0012, Japan.
authors: Shiori
authors: S
authors: Kawase
authors: Department of Molecular and Cellular Physiology, Institute of Medicine, Academic Assembly, Shinshu University, Nagano 390-8621, Japan.|CREST, JST, Saitama 332-0012, Japan.
authors: Rie
authors: R
authors: Koike
authors: Department of Molecular and Cellular Physiology, Institute of Medicine, Academic Assembly, Shinshu University, Nagano 390-8621, Japan.
authors: Michiru
authors: M
authors: Satoga
authors: Department of Molecular and Cellular Physiology, Institute of Medicine, Academic Assembly, Shinshu University, Nagano 390-8621, Japan.
authors: Xueshan
authors: X
authors: Cao
authors: Department of Molecular and Cellular Physiology, Institute of Medicine, Academic Assembly, Shinshu University, Nagano 390-8621, Japan.
authors: Xue
authors: X
authors: Li
authors: Department of Oral and Maxillofacial Surgery, Faculty of Medicine, University of Tsukuba, Ibaraki 305-8575, Japan.
authors: Toru
authors: T
authors: Yanagawa
authors: Department of Cardiovascular Research, Shinshu University Graduate School of Medicine, Nagano 390-8621, Japan.
authors: Takayuki
authors: T
authors: Sakurai
authors: Department of Cardiovascular Research, Shinshu University Graduate School of Medicine, Nagano 390-8621, Japan.
authors: Takayuki
authors: T
authors: Shindo
authors: Department of Molecular and Cellular Physiology, Institute of Medicine, Academic Assembly, Shinshu University, Nagano 390-8621, Japan.|Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Nagano 390-8621, Japan.|PRESTO, JST, Saitama 332-0012, Japan.
authors: Katsuhiko
authors: K
authors: Tabuchi
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D018274:Electroporation / Q000379:methods; D005260:Female; D015870:Gene Expression; D055879:Gene Knock-In Techniques / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008813:Mice, Inbred ICR; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D011247:Pregnancy; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000199:Actins; D009419:Nerve Tissue Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins