Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland.
Department of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, AL. 29 Listopada 54, 31-425 Krakow, Poland.
Int J Mol Sci. 2021 Mar 13;22(6):2937. doi: 10.3390/ijms22062937.
The 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.
Cas9 蛋白（SpCas9）是微生物中基于 CRISPR 的免疫系统的一个组成部分，已被广泛用于基因组编辑。这种核酸酶与指导 RNA（gRNA）形成核糖核蛋白（RNP）复合物，诱导 Cas9 结构变化并触发其切割活性。在这里，我们使用电子圆二色性（ECD）光谱来确认 RNP 的形成并确定其各个组成部分。ECD 光谱具有区分 Cas9 和 gRNA 的特征，前者显示出负/正轮廓，最大值位于 221、209 和 196nm，而后者显示出正/负/正/负模式，分别在 266、242、222 和 209nm 处出现带。首次提出了 gRNA:Cas9 RNP 复合物的实验 ECD 光谱。它表现出双信号正/负 ECD 偶联，最大值位于 273 和 235nm，与每个 RNP 成分的单个光谱明显不同。此外，Cas9 蛋白和 RNP 复合物在 ECD 测量后保留了生物活性，并且能够在体外结合和切割 DNA。因此，我们得出结论，ECD 光谱可以被认为是一种快速且非破坏性的方法，用于监测 Cas9 蛋白由于 Cas9 和 gRNA 相互作用而导致的构象变化，以及鉴定 gRNA:Cas9 RNP 复合物。
