Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Institute for Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Strasse 3a, 06120 Halle, Germany.
Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, 7 Saulėtekio Avenue, 10257 Vilnius, Lithuania.
J Am Soc Mass Spectrom. 2020 Mar 4;31(3):538-546. doi: 10.1021/jasms.9b00011. Epub 2020 Jan 30.
The adaptive immune system of bacteria and archaea against viral DNA is based on clustered, regularly interspaced, short palindromic repeats (CRISPRs) which are encoded in the host genome and translated into CRISPR RNAs (crRNAs) containing single spacer sequences complementary to foreign DNA. crRNAs assemble with CRISPR-associated (Cas) proteins forming surveillance complexes that base-pair with viral DNA and mediate its degradation. As specificity of degradation is provided by the crRNA spacer sequence, genetic engineering of the CRISPR system has emerged as a popular molecular tool, for instance, in gene silencing and programmed DNA degradation. Elongating or shortening the crRNA spacer sequence are therefore promising ventures to modify specificity toward the target DNA. However, even though the stoichiometry of wild-type complexes is well established, it is unknown how variations in crRNA spacer length affect their stoichiometry. The CRISPR-associated antiviral defense surveillance complexes of (StCascade complexes) contain crRNA and five protein subunits. Using native mass spectrometry, we studied the formation and stoichiometry of StCascade complexes assembled on a set of crRNAs with different spacer lengths. We assigned all relevant complexes and gained insights into the stoichiometry of the complexes as well as their preferred assembly. We found that stable complexes, which incorporate or lose a (Cas7)(Cse2)-module, assemble on crRNA varied in length by 12-nucleotide units, while varying crRNA length in six-nucleotide units results in heterogeneous mixtures of complexes. Combining our results from the various variants, we generated an assembly pathway revealing general features of I-E type Cascade complex formation.
细菌和古菌针对病毒 DNA 的适应性免疫系统基于成簇、规律间隔、短回文重复序列 (CRISPRs)，这些序列编码在宿主基因组中，并被翻译成含有与外源 DNA 互补的单一间隔序列的 CRISPR RNA (crRNA)。crRNA 与 CRISPR 相关 (Cas) 蛋白组装形成监测复合物，与病毒 DNA 碱基配对，并介导其降解。由于降解的特异性由 crRNA 间隔序列提供，因此 CRISPR 系统的基因工程已成为一种流行的分子工具，例如在基因沉默和程序性 DNA 降解中。因此，延长或缩短 crRNA 间隔序列是提高对目标 DNA 特异性的有前途的尝试。然而，尽管野生型复合物的化学计量比已经得到很好的确立，但尚不清楚 crRNA 间隔长度的变化如何影响它们的化学计量比。（StCascade 复合物）的 CRISPR 相关抗病毒防御监测复合物包含 crRNA 和五个蛋白质亚基。使用天然质谱法，我们研究了一组具有不同间隔长度的 crRNA 组装的 StCascade 复合物的形成和化学计量比。我们分配了所有相关的复合物，并深入了解了复合物的化学计量比及其优先组装。我们发现，能够结合或失去 (Cas7)(Cse2)-模块的稳定复合物可以在 crRNA 长度上变化 12 个核苷酸单位，而在 crRNA 长度上变化 6 个核苷酸单位则会导致复合物的混合物不均匀。结合我们从各种变体中获得的结果，我们生成了一个组装途径，揭示了 I-E 型 Cascade 复合物形成的一般特征。
