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

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.

The Physical Chemistry and Chemical Physics (PCCP) Section of the International Journal of Molecular Sciences in Its Publications: The First 300 Thematic Articles in the First 3 Years

“…In this Thematic Area, 56 articles have been published [ 48 , 64 , 104 , 108 , 110 , 124 , 195 , 252 , 253 , 254 , 255 , 256 , 257 , 258 , 259 , 260 , 261 , 262 , 263 , 264 , 265 , 266 , 267 , 268 , 269 , 270 , 271 , 272 , 273 , 274 , 275 , 276 , 277 , 278 , 279 , 280 , 281 , 282 , 283 , 284 , 285 , 286 , 287 , 288 , 289 , 290 , 291 , 292 , 293 , 294 , 295 , 296 , 297 , 298 , 299 , 300 ], an...…”

“…A more detailed examination shows that, in this direction, as well as in the previous ones, articles in which the objects of research are organic materials clearly prevail over articles in which the objects of research are inorganic materials (36 and 20, respectively). Wherein, the overwhelming majority of articles of the “organic” profile are, in one way or another, connected with the participation of the objects with a biological origin; these include [ 64 , 124 , 254 , 255 , 256 , 257 , 258 , 259 , 260 , 261 , 262 , 263 , 274 , 277 , 278 , 282 , 284 , 289 , 291 , 292 , 293 , 294 , 295 , 296 , 298 , 299 , 300 ] and the processes in abiogenic organic materials were considered only in [ 48 , 195 , 266 , 271 , 272 , 273 , 283 , 288 , 290 ]. The thematic focus of these works, as well as the chemical compounds studied in them, is also very diverse, and even a simple listing of them here is clearly beyond the scope of this article, too.…”

Physicochemical and Functional Characterization of Differential CRISPR-Cas9 Ribonucleoprotein Complexes

“…For instance, electronic circular dichroism (ECD) spectroscopy has been used to confirm Cas9 RNP complex formation and to detect individual components. 26 Although ECD and similar methods enable the monitoring of the conformational changes in Cas9 protein and Cas9 RNP complex formation, they are mainly qualitative and do not provide separation of each component. Traditional biochemical methods such as western blotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) can be used to confirm the results obtained from the spectroscopic methods, as well as to understand the biological significance of the Cas9 RNP complex in vivo.…”

“…Traditional biochemical methods such as western blotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) can be used to confirm the results obtained from the spectroscopic methods, as well as to understand the biological significance of the Cas9 RNP complex in vivo. 21,26,27 However, these techniques have drawbacks such as difficulty in quantification, low throughput, and manual handling steps. 28 High-performance liquid chromatography (HPLC) separation-based techniques are promising alternatives as they are quantitative and of high resolution 29 in the characterization of large molecules (i.e., 10−200 kDa).…”

“…Microscopy techniques such as X-ray, ,, cryo-transmission electron microscopy, , and spectroscopy methods − are commonly used for the structural characterization of the Cas9 protein and the Cas9 RNP complex. For instance, electronic circular dichroism (ECD) spectroscopy has been used to confirm Cas9 RNP complex formation and to detect individual components .…”