ABSTRACT

CRISPR type III systems, which are abundantly found in archaea, recognize and degrade RNA in their specific response to invading nucleic acids. Therefore, these systems can be harnessed for gene knockdown technologies even in hyperthermophilic archaea to study essential genes. We show here the broader usability of this posttranscriptional silencing technology by expanding the application to further essential genes and systematically analysing and comparing silencing thresholds and escape mutants. Synthetic guide RNAs expressed from miniCRISPR cassettes were used to silence genes involved in cell division (cdvA), transcription (rpo8), and RNA metabolism (smAP2) of the two crenarchaeal model organisms Saccharolobus solfataricus and Sulfolobus acidocaldarius. Results were systematically analysed together with those obtained from earlier experiments of cell wall biogenesis (slaB) and translation (aif5A). Comparison of over 100 individual transformants revealed gene-specific silencing maxima ranging between 40 and 75%, which induced specific knockdown phenotypes leading to growth retardation. Exceedance of this threshold by strong miniCRISPR constructs was not tolerated and led to specific mutation of the silencing miniCRISPR array and phenotypical reversion of cultures. In two thirds of sequenced reverted cultures, the targeting spacers were found to be precisely excised from the miniCRISPR array, indicating a still hypothetical, but highly active recombination system acting on the dynamics of CRISPR spacer arrays. Our results indicate that CRISPR type III – based silencing is a broadly applicable tool to study in vivo functions of essential genes in Sulfolobales which underlies a specific mechanism to avoid malignant silencing overdose.

 抽象的

CRISPR III 型系统在古细菌中大量存在，在对入侵核酸的特异性反应中识别并降解 RNA。因此，即使在超嗜热古细菌中，这些系统也可以用于基因敲除技术来研究必需基因。我们在这里通过将应用扩展到更多必需基因并系统地分析和比较沉默阈值和逃逸突变体，展示了这种转录后沉默技术的更广泛的可用性。从miniCRISPR盒表达的合成指导RNA被用来沉默涉及两种穹顶模型生物Saccharolobus solfataricus和Sulfolobus Acidocaldarius的细胞分裂（ cdvA ）、转录（ rpo8 ）和RNA代谢（ smAP2 ）的基因。结果与早期细胞壁生物发生 ( slaB ) 和翻译 ( aif5A ) 实验获得的结果一起进行了系统分析。对 100 多个个体转化体的比较揭示了基因特异性沉默最大值范围在 40% 至 75% 之间，这诱导了导致生长迟缓的特定敲低表型。强的 miniCRISPR 构建体超过这个阈值是不能容忍的，并导致沉默 miniCRISPR 阵列的特异性突变和培养物的表型逆转。在三分之二的测序恢复培养物中，发现靶向间隔区被精确地从 miniCRISPR 阵列中切除，这表明仍然是假设的，但高度活跃的重组系统作用于 CRISPR 间隔区阵列的动力学。 我们的结果表明，基于 CRISPR III 型的沉默是一种广泛适用的工具，可用于研究硫化叶菌中必需基因的体内功能，这是避免恶性沉默过量的特定机制的基础。