Laboratory of Nanoscale Biophysics and Biochemistry, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
Mol Cell. 2019 Jan 17;73(2):278-290.e4. doi: 10.1016/j.molcel.2018.11.008. Epub 2018 Nov 29.
Adaptive immune systems must accurately distinguish between self and non-self in order to defend against invading pathogens while avoiding autoimmunity. Type III CRISPR-Cas systems employ guide RNA to recognize complementary RNA targets, which triggers the degradation of both the invader's transcripts and their template DNA. These systems can broadly eliminate foreign targets with multiple mutations but circumvent damage to the host genome. To explore the molecular basis for these features, we use single-molecule fluorescence microscopy to study the interaction between a type III-A ribonucleoprotein complex and various RNA substrates. We find that Cas10-the DNase effector of the complex-displays rapid conformational fluctuations on foreign RNA targets, but is locked in a static configuration on self RNA. Target mutations differentially modulate Cas10 dynamics and tune the CRISPR interference activity in vivo. These findings highlight the central role of the internal dynamics of CRISPR-Cas complexes in self versus non-self discrimination and target specificity.
适应性免疫系统必须准确地区分自我和非自我，以抵御入侵病原体，同时避免自身免疫。III 型 CRISPR-Cas 系统利用指导 RNA 识别互补的 RNA 靶标，从而触发入侵者的转录本及其模板 DNA 的降解。这些系统可以广泛消除具有多种突变的外来靶标，但避免对宿主基因组造成损害。为了探索这些特性的分子基础，我们使用单分子荧光显微镜研究了 III-A 型核糖核蛋白复合物与各种 RNA 底物之间的相互作用。我们发现，该复合物的 DNA 酶效应子 Cas10 在 foreign RNA 靶标上表现出快速的构象波动，但在 self RNA 上则被锁定在静态构象中。靶标突变以不同的方式调节 Cas10 的动力学，并调节体内的 CRISPR 干扰活性。这些发现强调了 CRISPR-Cas 复合物内部动力学在自我与非自我区分和靶标特异性中的核心作用。
