PDF(Pubmed)
Abstract：
The introduction of CRISPR/Cas9 based gene editing has greatly accelerated therapeutic genome editing. However, the off-target DNA cleavage by CRISPR/Cas9 protein hampers its clinical translation, hindering its widespread use as a programmable genome editing tool. Although Cas9 variants with better mismatch discrimination have been developed, they have significantly lower rates of on-target DNA cleavage. Here, we have compared the dynamics of a more specific naturally occurring Cas9 from Francisella novicida (FnCas9) to the most widely used, SpCas9 protein. Long-scale atomistic MD simulation of free and gRNA bound forms of both the Cas9 proteins was performed, and their domain rearrangements and binding affinity with gRNA were compared to decipher the possible reason behind the enhanced specificity of FnCas9 protein. The greater binding affinity with gRNA, high domain electrostatics, and more volatility of FnCas9 than SpCas9 may explain its increased specificity and lower tolerance for mismatches.
摘要：
基于CRISPR/Cas9的基因编辑的引入极大地加速了治疗性基因组编辑。然而,CRISPR/Cas9蛋白的脱靶DNA切割阻碍了其临床翻译，阻碍了其作为可编程基因组编辑工具的广泛使用。尽管已经开发了具有更好的错配区分的Cas9变体，它们具有显著较低的靶DNA切割率。这里,我们已经比较了一个更具体的自然发生的Cas9的动力学从Francisellanovicida（FnCas9）最广泛使用，SpCas9蛋白。进行了两种Cas9蛋白的游离和gRNA结合形式的长期原子MD模拟，与它们的结构域重排和与gRNA的结合亲和力进行比较，以破译FnCas9蛋白特异性增强背后的可能原因。与gRNA的结合亲和力越大，高域静电，FnCas9比SpCas9更多的波动性可能解释了其特异性增加和对错配的耐受性较低。
