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

• Published in: PloS one 2015-07, Vol.10 (7), p.e0132363
• Main Authors: Tsukamoto, Kentaro, Ozeki, Chikako, Kohda, Tomoko, Tsuji, Takao
• Format: Article
• Language: English
• Subjects: Animals; beta-D-Galactoside alpha 2-6-Sialyltransferase; Biological activity; Botulinum toxin type C; Botulinum Toxins - toxicity; Botulism; Cell Line, Tumor; Cell Proliferation - drug effects; Clonal deletion; Clostridium botulinum; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; CRISPR; Embryonal Carcinoma Stem Cells - cytology; Embryonal Carcinoma Stem Cells - metabolism; Gangliosides; Gangliosides - pharmacology; Gene deletion; Gene expression; Genomes; Intracellular; Mice; Microscopy, Confocal; N-acetylgalactosaminyltransferase; N-Acetylgalactosaminyltransferases - antagonists & inhibitors; N-Acetylgalactosaminyltransferases - deficiency; N-Acetylgalactosaminyltransferases - genetics; N-Acetylgalactosaminyltransferases - metabolism; Nerve endings; Neurogenesis; Neurons; Neurotoxins; Neurotransmitter release; Paralysis; Polypeptide N-acetylgalactosaminyltransferase; Proteins; Proteolysis - drug effects; RNA Interference; RNA, Small Interfering - metabolism; Sensitivity; Sialyltransferases - antagonists & inhibitors; Sialyltransferases - genetics; Sialyltransferases - metabolism; Stem cells; Surface Plasmon Resonance; Toxins
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0132363

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.