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

Jagoda Orleanska,Rafal Baranski,Magdalena Klimek-Chodacka,Monika Halat,Malgorzata Baranska

doi:10.3390/ijms22062937

Abstract

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.

Highlights

In 2020, the Royal Swedish Academy of Sciences has awarded the Nobel Prize in Chemistry for the development of a method for genome editing, as the consequence of discovery and research on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins
We found that electronic circular dichroism (ECD) can be considered as a quick and effective method to identify the guide RNA (gRNA):Cas9 RNP complex
Despite a lot of ECD studies describing protein—nucleic acids interactions [55,56], here we present for the first time the experimental ECD spectrum of gRNA:Cas9 RNP complex (Figure 4, red line)

Summary

Introduction

In 2020, the Royal Swedish Academy of Sciences has awarded the Nobel Prize in Chemistry for the development of a method for genome editing, as the consequence of discovery and research on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins. A wide range of potential applications and the simplicity, precision and effectiveness of genome editing justified the earlier announcement of CRISPR/Cas the Breakthrough of the Year by Science journal in 2015 [2]. The CRISPR/Cas-based methods have been revolutionizing biological sciences, nowadays. Advanced research has been carried out for the application of CRISPR/Cas in human disease treatments, including AIDS, hemophilia, autism spectrum disorder, and cancer [5].

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Conclusion