Next Article in Journal
3D Environment Is Required In Vitro to Demonstrate Altered Bone Metabolism Characteristic for Type 2 Diabetics
Next Article in Special Issue
Ring-Selective Fragmentation in the Tirapazamine Molecule upon Low-Energy Electron Attachment
Previous Article in Journal
Overexpression of AtBBD1, Arabidopsis Bifunctional Nuclease, Confers Drought Tolerance by Enhancing the Expression of Regulatory Genes in ABA-Mediated Drought Stress Signaling
Previous Article in Special Issue
Elastic Electron Scattering from Methane Molecule in the Energy Range from 50–300 eV
 
 
Article

Electronic Circular Dichroism of the Cas9 Protein and gRNA:Cas9 Ribonucleoprotein Complex

1
Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland
2
Department of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, AL. 29 Listopada 54, 31-425 Krakow, Poland
3
Jagiellonian Centre for Experimental Therapeutics (JCET), Bobrzyńskiego 14, 30-348 Krakow, Poland
*
Authors to whom correspondence should be addressed.
Academic Editors: Filipe Ferreira da Silva and François Hache
Int. J. Mol. Sci. 2021, 22(6), 2937; https://doi.org/10.3390/ijms22062937
Received: 15 February 2021 / Revised: 9 March 2021 / Accepted: 11 March 2021 / Published: 13 March 2021
(This article belongs to the Special Issue Electron and Photon Interactions with Bio(Related) Molecules)
The Streptococcus pyogenes Cas9 protein (SpCas9), a component of CRISPR-based immune system in microbes, has become commonly utilized for genome editing. This nuclease forms a ribonucleoprotein (RNP) complex with guide RNA (gRNA) which induces Cas9 structural changes and triggers its cleavage activity. Here, electronic circular dichroism (ECD) spectroscopy was used to confirm the RNP formation and to determine its individual components. The ECD spectra had characteristic features differentiating Cas9 and gRNA, the former showed a negative/positive profile with maxima located at 221, 209 and 196 nm, while the latter revealed positive/negative/positive/negative pattern with bands observed at 266, 242, 222 and 209 nm, respectively. For the first time, the experimental ECD spectrum of the gRNA:Cas9 RNP complex is presented. It exhibits a bisignate positive/negative ECD couplet with maxima at 273 and 235 nm, and it differs significantly from individual spectrum of each RNP components. Additionally, the Cas9 protein and RNP complex retained biological activity after ECD measurements and they were able to bind and cleave DNA in vitro. Hence, we conclude that ECD spectroscopy can be considered as a quick and non-destructive method of monitoring conformational changes of the Cas9 protein as a result of Cas9 and gRNA interaction, and identification of the gRNA:Cas9 RNP complex. View Full-Text
Keywords: CRISPR/Cas9; DNA cleavage; ECD; spectroscopy; guide RNA; RNP complex; SpCas9 CRISPR/Cas9; DNA cleavage; ECD; spectroscopy; guide RNA; RNP complex; SpCas9
Show Figures

Figure 1

MDPI and ACS Style

Halat, M.; Klimek-Chodacka, M.; Orleanska, J.; Baranska, M.; Baranski, R. Electronic Circular Dichroism of the Cas9 Protein and gRNA:Cas9 Ribonucleoprotein Complex. Int. J. Mol. Sci. 2021, 22, 2937. https://doi.org/10.3390/ijms22062937

AMA Style

Halat M, Klimek-Chodacka M, Orleanska J, Baranska M, Baranski R. Electronic Circular Dichroism of the Cas9 Protein and gRNA:Cas9 Ribonucleoprotein Complex. International Journal of Molecular Sciences. 2021; 22(6):2937. https://doi.org/10.3390/ijms22062937

Chicago/Turabian Style

Halat, Monika, Magdalena Klimek-Chodacka, Jagoda Orleanska, Malgorzata Baranska, and Rafal Baranski. 2021. "Electronic Circular Dichroism of the Cas9 Protein and gRNA:Cas9 Ribonucleoprotein Complex" International Journal of Molecular Sciences 22, no. 6: 2937. https://doi.org/10.3390/ijms22062937

Find Other Styles
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop