Immunogold Labeling to Detect Streptococcus pyogenes Cas9 in Cell Culture and Tissues by Electron Microscopy

• Published in: CRISPR journal Vol. 2; no. 6; p. 395
• Main Authors: Ulloa-Navas, María José, García-Tárraga, Patricia, García-Verdugo, José Manuel, Herranz-Pérez, Vicente
• Format: Journal Article
• Language: English
• Published: United States 01.12.2019
• Subjects: Animals; Brain; Clustered Regularly Interspaced Short Palindromic Repeats - immunology; CRISPR-Associated Protein 9 - genetics; CRISPR-Cas Systems - immunology; DNA; Gene Editing - methods; Genetic Vectors; HEK293 Cells; Humans; Mice; Mice, Inbred C57BL; Microscopy, Electron - methods; Microscopy, Immunoelectron - methods; RNA, Guide, CRISPR-Cas Systems; Streptococcus pyogenes - genetics
• ISSN: 2573-1602
• DOI: 10.1089/crispr.2019.0032

The CRISPR-Cas9 system is a powerful and yet precise DNA-editing tool in rapid development. By combining immunogold labeling and electron microscopy with the novel CRISPR-Cas9 system, we propose a new method to gain insight into the biology of this tool. In this study, we analyzed different Cas9-induced systems such as HEK293T cell line, murine oligodendrocyte progenitor cells, brain and liver to detect Cas9 expression by immunoelectron microscopy. Our results show that while Cas9 expression could be found in the nuclei and nucleopores of transfected HEK293T cells, in transfected oligodendrocyte precursor cells, Cas9 was found in cytoplasmic vesicles. In Cas9 constitutively expressing oligodendrocyte precursors, the enzyme was located in the cytoplasm of nondividing cells. Finally, while in the liver Cas9 was detected in different cell types, in the brain we found no specifically labeled cells. In conclusion, immunoelectron microscopy opens a new spectrum of opportunities to study the CRISPR-Cas9 system in a more precise manner.