Biology subjects

Fehrenbach, A.

Publications and source records attributed to Fehrenbach, A..

3 recordsLinked to original sources

bioRxiv2025

An evolutionary approach to predict the orientation of CRISPR arrays

CRISPR-Cas is a defense system of bacteria and archaea against phages. Parts of the foreign DNA, called spacers, are incorporated into the CRISPR array which constitutes the immune memory. The orientation of CRISPR arrays is crucial for analyzing and understanding the functionality of CRISPR systems and their targets. Several methods have been developed to identify the orientation of a CRISPR array. To predict the orientation, different methods use different features such as the repeat sequences between the spacers, the location of the leader sequence, the Cas genes, or PAMs. However, those features are often not sufficient to predict the orientation with certainty, or different methods disagree. Remarkably, almost all CRISPR systems have been found to insert spacers in a polarized manner at the leader end of the array. We introduce CRISPR-evOr, a method that leverages the resulting patterns to predict the acquisition orientation for (a group of) CRISPR arrays by reconstructing and comparing the likelihood of their evolutionary history with respect to both possible acquisition orientations. The new method is independent of Cas type, leader existence and location, and transcription orientation. CRISPR-evOr is thus particularly useful for arrays that other CRISPR orientation tools cannot predict confidently and to verify or resolve conflicting predictions from existing tools. CRISPR-evOr currently confidently predicts the orientation of 28.3% of the arrays in the considered subset of CRISPRCasdb, which other tools like CRISPRDirection and CRISPRstrand cannot reliably orient. As our tool leverages evolutionary information we expect this percentage to grow in the future when more closely related arrays will be available. Additionally, CRISPR-evOr provides confident decisions for rare subtypes of CRISPR arrays, where knowledge about repeats and leaders and their orientation is limited. Author SummarySome bacteria and archaea possess a CRISPR-Cas defense system, which protects them against phages and mobile genetic elements. This system adapts to new threats by incorporating small fragments of their DNA, so called spacers, in a CRISPR array. Remarkably, the acquisition of new spacers is polarized at one end of this array. To understand how this immune system functions, it is essential to know the orientation of these arrays. Many existing tools try to determine orientation using genetic markers, but these methods are often unreliable or disagree with one another. In our work, we developed a new method that predicts the end at which new spacers are inserted, by considering the evolutionary history of a group of related CRISPR arrays. Unlike other tools, our approach is less reliant on specific genetic markers and can be applied broadly across many types of CRISPR systems. We show that it can confidently determine the orientation of a large number of arrays that other methods cannot resolve. This provides a new way to predict the array orientation, which is particularly useful for rare CRISPR types. Our evolutionary approach will become even more powerful as more genetic data becomes available.

bioinformatics↗

bioRxiv2025

The complexity of multiple CRISPR arrays in strains with (co-occurring) CRISPR systems

CRISPR and their associated Cas proteins provide adaptive immunity in prokaryotes, protecting against invading genetic elements. These systems are categorized into types and are highly diverse. Genomes often harbor multiple CRISPR arrays varying in length and distance from Cas loci. However, the ecological roles of multiple CRISPR arrays and their interactions with multiple Cas loci remain poorly understood. We present a comprehensive analysis of CRISPR systems that uncovers variation between diverse Cas types regarding the occurrence of multiple arrays, the distribution of their lengths and positions relative to Cas loci, and the diversity of their repeat sequences. Some types tend to occur as the sole Cas locus present in the genome, but typically have two or more associated arrays, especially for types I-E and I-F. Multiple Cas types are also common, with some systems showing a preference for specific co-occurrence. Distinct array distributions and orientations around Cas loci indicate substantial differences in functionality and transcriptional behavior among Cas types. Our analysis suggests that arrays with identical repeats in the same genome acquire new spacers at comparable rates, irrespective of their proximity to the Cas locus. Furthermore, repeat similarities indicate that arrays of systems that often co-occur with other systems tend to have more diverse repeats than those mostly appearing alongside solitary systems. Our results indicate that co-occurring Cas type pairs might not only collaborate in spacer acquisition but also maintain independent and complementary functions and that CRISPR systems distribute their defensive spacer repertoire equally across multiple CRISPR arrays.

microbiology↗

bioRxiv2024

SpacerPlacer: Ancestral reconstruction of CRISPR arrays reveals the evolutionary dynamics of spacer deletions

Bacteria employ CRISPR-Cas systems for defense by integrating invader-derived sequences, termed spacers, into the CRISPR array, which constitutes an immunity memory. While spacer deletions occur randomly across the array, newly acquired spacers are predominantly integrated at the leader end. Consequently, spacer arrays can be used to derive the chronology of spacer insertions. Reconstruction of ancestral spacer acquisitions and deletions could help unravel the coevolution of phages and bacteria, the evolutionary dynamics in microbiomes, or track pathogens. However, standard reconstruction methods produce misleading results by overlooking insertion order and joint deletions of spacers. Here, we present SpacerPlacer, a maximum likelihood-based ancestral reconstruction approach for CRISPR array evolution. We used SpacerPlacer to reconstruct and investigate ancestral deletion events of 4565 CRISPR arrays, revealing that spacer deletions occur 374 times more frequently than mutations and are regularly deleted jointly, with an average of 2.7 spacers. Surprisingly, we observed a decrease in the spacer deletion frequency towards both ends of the reconstructed arrays. While the resulting trailer-end conservation is commonly observed, a reduced deletion frequency is now also detectable towards the variable leader end. Finally, our results point to the hypothesis that frequent loss of recently acquired spacers may provide a selective advantage.

bioinformatics↗