Transposable Elements in the Genome of the Lichen-Forming Fungus Umbilicaria pustulata and Their Distribution in Different Climate Zones along Elevation

Transposable elements (TEs) are an important source of genome plasticity across the tree of life. Drift and natural selection are important forces shaping TE distribution and accumulation. Fungi, with their multifaceted phenotypic diversity and relatively small genome size, are ideal models to study the role of TEs in genome evolution and their impact on the host’s ecological and life history traits. Here we present an account of all TEs found in a high-quality reference genome of the lichen-forming fungus Umbilicaria pustulata, a macrolichen species comprising two climatic ecotypes: Mediterranean and cold temperate. We trace the occurrence of the newly identified TEs in populations along three elevation gradients using a Pool-Seq approach to identify TE insertions of potential adaptive significance. We found that TEs cover 21.26% of the 32.9 Mbp genome, with LTR Gypsy and Copia clades being the most common TEs. We identified 28 insertions displaying consistent insertion frequency differences between the two host ecotypes across the elevation gradients. Most of the highly differentiated insertions were located near genes, indicating a putative function. This pioneering study of the content and climate niche-specific distribution of TEs in a lichen-forming fungus contributes to understanding the roles of TEs in fungal evolution.

Predicting efficiency of writing short sequences into the genome using prime editing

Any short sequence can be precisely written into a selected genomic target using prime editing. This ability facilitates protein tagging, correction of pathogenic deletions, and many other exciting applications. However, it remains unclear what types of sequences prime editors can efficiently insert, and how to choose optimal reagents for a desired outcome. To characterize features that influence insertion efficiency, we designed a library of 2,666 sequences up to 69 nt in length and measured the frequency of their insertion into four genomic sites in three human cell lines, using different prime editor systems. We discover that insertion sequence length, nucleotide composition and secondary structure all affect insertion rates, and that mismatch repair proficiency is a strong determinant for the shortest insertions. Combining the sequence and repair features into a machine learning model, we can predict insertion frequency for new sequences with R = 0.69. The tools we provide allow users to choose optimal constructs for DNA insertion using prime editing.

Small molecule inhibition of ATM kinase increases CRISPR-Cas9 1-bp insertion frequency

Mutational outcomes following CRISPR-Cas9-nuclease cutting in mammalian cells have recently been shown to be predictable and, in certain cases, skewed toward single genotypes. However, the ability to control these outcomes remains limited, especially for 1-bp insertions, a common and therapeutically relevant class of repair outcomes. Here, through a small molecule screen, we identify the ATM kinase inhibitor KU-60019 as a compound capable of reproducibly increasing the fraction of 1-bp insertions relative to other Cas9 repair outcomes. Small molecule or genetic ATM inhibition increases 1-bp insertion outcome fraction across three human and mouse cell lines, two Cas9 species, and dozens of target sites, although concomitantly reducing the fraction of edited alleles. Notably, KU-60019 increases the relative frequency of 1-bp insertions to over 80% of edited alleles at several native human genomic loci and improves the efficiency of correction for pathogenic 1-bp deletion variants. The ability to increase 1-bp insertion frequency adds another dimension to precise template-free Cas9-nuclease genome editing.

Transposable elements in the genome of the lichen-forming fungus Umbilicaria pustulata, and their distribution in different climate zones along elevation

Background: Transposable elements (TEs) are an important source of genome plasticity across the tree of life. Accumulating evidence suggests that TEs may not be randomly distributed in the genome. Drift and natural selection are important forces shaping TE distribution and accumulation, acting directly on the TE element or indirectly on the host species. Fungi, with their multifaceted phenotypic diversity and relatively small genome size, are ideal models to study the role of TEs in genome evolution and their impact on the host's ecological and life history traits. Here we present an account of all TEs found in a high-quality reference genome of the lichen-forming fungus Umbilicaria pustulata, a macrolichen species comprising two climatic ecotypes: Mediterranean and cold-temperate. We trace the occurrence of the newly identified TEs in populations along three replicated elevation gradients using a Pool-Seq approach, to identify TE insertions of potential adaptive significance. Results: We found that TEs cover 21.26 % of the 32.9 Mbp genome, with LTR Gypsy and Copia clades being the most common TEs. Out of a total of 182 TE copies we identified 28 insertions displaying consistent insertion frequency differences between the two host ecotypes across the elevation gradients. Most of the highly differentiated insertions were located near genes, indicating a putative function. Conclusions: This pioneering study into the content and climate niche-specific distribution of TEs in a lichen-forming fungus contributes to understanding the roles of TEs in fungal evolution. Particularly, it may serve as a foundation for assessing the impact of TE dynamics on fungal adaptation to the abiotic environment, and the impact of TE activity on the evolution and maintenance of a symbiotic lifestyle.

Removal of beneficial insertion effects prevent the long-term persistence of transposable elements within simulated asexual populations

Background Transposable elements are significant components of most organism’s genomes, yet the reasons why their abundances vary significantly among species is poorly understood. A recent study has suggested that even in the absence of traditional molecular evolutionary explanations, transposon proliferation may occur through a process known as ‘transposon engineering’. However, their model used a fixed beneficial transposon insertion frequency of 20%, which we believe to be unrealistically high. Results Reducing this beneficial insertion frequency, while keeping all other parameters identical, prevented transposon proliferation. Conclusions We conclude that the author’s original findings are better explained through the action of positive selection rather than ‘transposon engineering’, with beneficial insertion effects remaining important during transposon proliferation events.

Transposable element dynamics are consistent across the Drosophila phylogeny, despite drastically differing content

BackgroundThe evolutionary dynamics of transposable elements (TEs) vary across the tree of life and even between closely related species with similar ecologies. In Drosophila, most of the focus on TE dynamics has been completed in Drosophila melanogaster and the overall pattern indicates that TEs show an excess of low frequency insertions, consistent with their frequent turn over and high fitness cost in the genome. Outside of D. melanogaster, insertions in the species Drosophila algonquin, suggests that this situation may not be universal, even within Drosophila. Here we test whether the pattern observed in D. melanogaster is similar across five Drosophila species that share a common ancestor more than fifty million years ago.ResultsFor the most part, TE family and order insertion frequency patterns are broadly conserved between species, supporting the idea that TEs have invaded species recently, are mostly costly and dynamics are conserved in orthologous regions of the host genomeConclusionsMost TEs retain similar activities and fitness costs across the Drosophila phylogeny, suggesting little evidence of drift in the dynamics of TEs across the phylogeny, and that most TEs have invaded species recently.

σB Regulates IS256-Mediated Staphylococcus aureus Biofilm Phenotypic Variation

ABSTRACT Biofilm formation in Staphylococcus aureus is subject to phase variation, and biofilm-negative derivatives emerge sporadically from a biofilm-positive bacterial population. To date, the only known mechanism for generating biofilm phenotypic variation in staphylococci is the reversible insertion/excision of IS256 in biofilm-essential genes. In this study, we present evidence suggesting that the absence of the σB transcription factor dramatically increases the rate of switching to the biofilm-negative phenotype in the clinical isolate S. aureus 15981, under both steady-state and flow conditions. The phenotypic switching correlates with a dramatic increase in the number of IS256 copies in the chromosomes of biofilm-negative variants, as well as with an augmented IS256 insertion frequency into the icaC and the sarA genes. IS256-mediated biofilm switching is reversible, and biofilm-positive variants could emerge from biofilm-negative σB mutants. Analysis of the chromosomal insertion frequency using a recombinant IS256 element tagged with an erythromycin marker showed an almost three-times-higher transposition frequency in a ΔσB strain. However, regulation of IS256 activity by σB appears to be indirect, since transposase transcription is not affected in the absence of σB and IS256 activity is inhibited to wild-type levels in a ΔσB strain under NaCl stress. Overall, our results identify a new role for σB as a negative regulator of insertion sequence transposition and support the idea that deregulation of IS256 activity abrogates biofilm formation capacity in S. aureus.