title: CRISPR/Cas9-Mediated Genomic Deletion of the Beta-1, 4 N-acetylgalactosaminyltransferase 1 Gene in Murine P19 Embryonal Carcinoma Cells Results in Low Sensitivity to Botulinum Neurotoxin Type C.
abstract: Botulinum neurotoxins produced by Clostridium botulinum cause flaccid paralysis by inhibiting neurotransmitter release at peripheral nerve terminals. Previously, we found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C. In order to prove the utility of P19 cells for the study of the intracellular mechanism of botulinum neurotoxins, ganglioside-knockout neurons were generated by deletion of the gene encoding beta-1,4 N-acetylgalactosaminyltransferase 1 in P19 cells using the clustered regularly interspaced short palindromic repeats combined with Cas9 (CRISPR/Cas9) system. By using this system, knockout cells could be generated more easily than with previous methods. The sensitivity of the generated beta-1,4 N-acetylgalactosaminyltransferase 1-depleted P19 neurons to botulinum neurotoxin type C was decreased considerably, and the exogenous addition of the gangliosides GD1a, GD1b, and GT1b restored the susceptibility of P19 cells to botulinum neurotoxin type C. In particular, addition of a mixture of these three ganglioside more effectively recovered the sensitivity of knockout cells compared to independent addition of GD1a, GD1b, or GT1b. Consequently, the genome-edited P19 cells generated by the CRISPR/Cas9 system were useful for identifying and defining the intracellular molecules involved in the toxic action of botulinum neurotoxins.
authors.affiliation: Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Tsukamoto
authors.affiliation: Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.
authors.forename: Chikako
authors.initials: C
authors.lastname: Ozeki
authors.affiliation: Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kohda
authors.affiliation: Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Tsuji
mesh_terms: D000818:Animals; D001905:Botulinum Toxins / Q000633:toxicity*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D054278:Embryonal Carcinoma Stem Cells / Q000166:cytology / Q000378:metabolism; D005732:Gangliosides / Q000494:pharmacology; D051379:Mice; D018613:Microscopy, Confocal; D017350:N-Acetylgalactosaminyltransferases / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D059748:Proteolysis / Q000187:drug effects; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D012799:Sialyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D001905:Botulinum Toxins; D005732:Gangliosides; D017350:N-Acetylgalactosaminyltransferases; D034741:RNA, Small Interfering; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C021298:ganglioside, GD1a; C033851:ganglioside, GD1b; C016481:trisialoganglioside GT1; C096324:botulinum toxin type C