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.
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