Clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas)9 transactivating CRISPR RNAs (tracrRNAs) form distinct structures essential for target recognition and cleavage and dictate exchangeability between orthologous proteins. As noncoding RNAs that are often apart from the CRISPR array, their identification can be arduous. In this article, a new bioinformatic method for the detection of Cas9 tracrRNAs is presented. The approach utilizes a covariance model based on both sequence homology and predicted secondary structure to locate tracrRNAs. This method predicts a tracrRNA for 98% of CRISPR-Cas9 systems identified by us. To ensure accuracy, we also benchmark our approach against biochemically vetted tracrRNAs finding false-positive and false-negative rates of 5.5% and 7.1%, respectively. Finally, the association between Cas9 amino acid sequence-based phylogeny and tracrRNA secondary structure is evaluated, revealing strong evidence that secondary structure is evolutionarily conserved among Cas9 lineages. Altogether, our findings provide insight into Cas9 tracrRNA evolution and efforts to characterize the tracrRNA of Cas9 systems.

中文翻译：

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Cas9 tracrRNA 的鉴定和进化


成簇的规则间隔回文重复序列 (CRISPR) 相关 (Cas)9 反式激活 CRISPR RNA (tracrRNA) 形成对于靶标识别和切割至关重要的独特结构，并决定直系同源蛋白质之间的可交换性。由于非编码 RNA 通常不存在于 CRISPR 阵列中，因此它们的鉴定可能非常困难。在本文中，提出了一种检测 Cas9 tracrRNA 的新生物信息学方法。该方法利用基于序列同源性和预测二级结构的协方差模型来定位 tracrRNA。该方法可以预测我们识别的 98% CRISPR-Cas9 系统的 tracrRNA。为了确保准确性，我们还将我们的方法与经过生化审查的 tracrRNA 进行基准测试，发现假阳性率和假阴性率分别为 5.5% 和 7.1%。最后，评估了基于 Cas9 氨基酸序列的系统发育与 tracrRNA 二级结构之间的关联，揭示了二级结构在 Cas9 谱系中进化上保守的有力证据。总而言之，我们的研究结果提供了对 Cas9 tracrRNA 进化的深入了解以及表征 Cas9 系统 tracrRNA 的努力。