Biology subjects

Schiffthaler, B.

Publications and source records attributed to Schiffthaler, B..

9 recordsLinked to original sources

bioRxiv2026

Single-Cell and Tissue-Specific CRISPR Editing Analyses Unveil New Insights to Off-Targets and Translocations

CRISPR-Cas9 holds promise for treating genetic disease, but rare off-target mutations and structural variants remain as key safety concerns, especially at scales relevant to therapy. We established workflows to resolve Cas9 off-target activity in vitro at single-cell resolution and in vivo across different tissues. Using clonally expanded electroporated mouse embryos and embryonic stem cells, we reveal that individual cells exhibit unique off-target and translocation profiles, including events missed in bulk analyses. Integrating single-cell editing with chromatin accessibility, transcription, and DNA methylation measurements suggested that sequence-independent features modulate Cas9 access and cleavage, with preferential editing in regions characterized by open chromatin and lower methylation. In Cas9-inducible mouse models, editing analyses revealed organ-distinct off-target spectra, DNA repair pathway usage, indel patterns, and markedly varying translocation propensity between tissues. These findings demonstrate that off-target activity is heterogeneous across cells and context-dependent across organs, motivating sensitive single-cell analyses and organ-specific evaluation in preclinical development to more accurately assess risk and improve the safety of CRISPR-based genomic medicines.

genomics↗

bioRxiv2024

Essentiality of LD-Transpeptidation in Agrobacterium tumefaciens

Peptidoglycan (PG), a mesh-like structure which is the primary component of the bacterial cell wall, is crucial to maintain cell integrity and shape. While most bacteria rely on penicillin binding proteins (PBPs) for crosslinking, some species employ LD-transpeptidases (LDTs). Unlike PBPs, the essentiality and biological functions of LDTs remain largely unclear. The Hyphomicrobiales order of the Alphaproteobacteria, known for their polar growth, have PG which is unusually rich in LD-crosslinks, suggesting that LDTs may play a more significant role in PG synthesis in these bacteria. Here, we investigated LDTs in the plant pathogen Agrobacterium tumefaciens and found that LD-transpeptidation, resulting from at least one of 14 putative LDTs present in this bacterium, is essential for its survival. Notably, a mutant lacking a distinctive group of 7 LDTs which are broadly conserved among the Hyphomicrobiales exhibited reduced LD-crosslinking and tethering of PG to outer membrane {beta}-barrel proteins. Consequently, this mutant suffered severe fitness loss and cell shape rounding, underscoring the critical role played by these Hyphomicrobiales-specific LDTs in maintaining cell wall integrity and promoting elongation. Tn-sequencing screens further revealed non-redundant functions for A. tumefaciens LDTs. Specifically, Hyphomicrobiales-specific LDTs exhibited synthetic genetic interactions with division and cell cycle proteins, and a single LDT from another group. Additionally, our findings demonstrate that strains lacking all LDTs except one displayed distinctive phenotypic profiles and genetic interactions. Collectively, our work emphasizes the critical role of LD-crosslinking in A. tumefaciens cell wall integrity and growth and provides insights into the functional specialization of these crosslinking activities.

microbiology↗

bioRxiv2022

Simultaneous inhibition of DNA-PK and Pol{Theta} improves integration efficiency and precision of genome editing

Genome editing tools, especially CRISPR/Cas9-based strategies, have transformed biomedical research and opened opportunities for developing curative treatments for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of undesired mutations represent major limitations for genome editing applications. Both issues arise from the interplay between the main DNA Double-Strand Break (DSB) repair pathways, Homology-Directed Repair (HDR), Non-Homologous End Joining (NHEJ), and Microhomology-Mediated End Joining (MMEJ). To improve efficiencies of targeted CRISPR-Cas9 genome editing, we screened a large compound library. This led to the discovery of AZD7648, a DNA-dependent protein kinase (DNA-PK) inhibitor and potent enhancer of CRISPR-Cas9-mediated integration. We demonstrated that AZD7648 increased HDR and decreased mutagenic NHEJ repair, thus resulting in improved performance of precise gene editing. Furthermore, we observed additional improvement of integration efficiency by impairing MMEJ repair through DNA polymerase {ominus} (Pol{ominus}) inhibition. Combined treatment with AZD7648 and Pol{ominus} inhibitors (which we named 2iHDR) substantially increased precision of templated insertions, with efficiencies of up to 80%, and nearly no formation of undesired Insertion-Deletions (InDels). Importantly, 2iHDR also decreased Cas9-associated off-target activity, dramatically improving the performance and fidelity of CRISPR-Cas9 gene editing.

molecular biology↗

bioRxiv2022

Genome-wide peptidoglycan profiling of Vibrio cholerae

Most bacteria cells are protected by a peptidoglycan cell wall. Defining the chemical structure of the peptidoglycan has been instrumental to characterize cell wall associated proteins and to illuminate the mode of action of cell wall-acting antibiotics. However, a major roadblock for a comprehensive understanding of peptidoglycan homeostasis has been the lack of methods to conduct large-scale, systematic studies. Here we have developed and applied an innovative high throughput peptidoglycan analytical pipeline to analyze the entire non-essential, arrayed mutant library of Vibrio cholerae. The unprecedented breadth of these analyses revealed that peptidoglycan homeostasis is preserved by a large percentage of the genome organized in complex networks that functionally link peptidoglycan features with genetic determinants. As an example, we discovered a novel bifunctional penicillin-binding protein in V. cholerae. Collectively, genome-wide peptidoglycan profiling provides a fast, easy, and unbiased method for systematic identification of the genetic determinants of peptidoglycan synthesis and remodeling.

microbiology↗

bioRxiv2019

An Improved Genome Assembly of the European Aspen Populus tremula

Aspen (Populus tremula L.) is a widely distributed keystone species and a model system for forest tree genomics, with extensive resources developed for population genetics and genomics. Here we present an updated resource comprising a chromosome-scale assembly of P. tremula and population genetics and genomics data integrated into the PlantGenIE.org web resource. We demonstrate use of the diverse data types included to explore the genetic basis of natural variation in leaf size and shape as examples of traits with complex genetic architecture. We present a chromosome-scale genome assembly generated using long-read sequencing, optical and high-density genetic maps containing 39,894 annotated genes with functional annotations for 73,765 transcripts from 37,184 gene loci. We conducted whole-genome resequencing of the Ume[a] Aspen (UmAsp) collection comprising 227 aspen individuals. We utilised the assembly, the UmAsp re-sequencing data and existing whole genome re-sequencing data from the Swedish Aspen (SwAsp) and Scottish Aspen (ScotAsp) collections to perform genome-wide association analyses (GWAS) using Single Nucleotide Polymorphisms (SNPs) for leaf physiognomy phenotypes. We conducted Assay of Transposase Accessible Chromatin sequencing (ATAC-Seq) and identified genomic regions of accessible chromatin and subset SNPs to these regions, which improved the GWAS detection rate. We identified candidate long non-coding RNAs in leaf samples and quantified their expression in an updated co-expression network (AspLeaf, available in PlantGenIE.org), which we further used to explore the functions of candidate genes identified from the GWAS. We examined synteny to the reference P. trichocarpa assembly and identified P. tremula-specific regions. Analysis of whole-genome duplication indicated differential substitution rates for the two Populus species, indicating more rapid evolution in P. tremula. A GWAS of 26 leaf physiognomy traits and all SNPs in each of the three aspen collections found significant associations for only two traits in ScotAsp collection and one in UmAsp, whereas subsetting SNPs to those in open chromatin regions revealed associations for a further four traits among all three aspen collections. The significant SNPs were associated with genes annotated for developmental and growth functions, which represent candidates for further study. Of particular interest was a 177-kbp region of chromosome 9 harbouring SNPs associated with multiple leaf phenotypes in ScotAsp, with the set of SNPs in linkage disequilibrium explaining 24 to 30 % of the phenotypic variation in leaf indent depth variation. We have incorporated the assembly, population genetics, genomics and leaf physiognomy GWAS data into the PlantGenIE.org web resource, including updating existing genomics data to the new genome version. This enables easy exploration and visualisation of the genomics data and exploration of GWAS results. We provide all raw and processed data used for the presented analyses to facilitate reuse in future studies.

genomics↗

bioRxiv2019

The mitogenome of Norway spruce and a reappraisal of mitochondrial recombination in plants

Plant mitogenomes can be difficult to assemble because they are structurally dynamic and prone to intergenomic DNA transfers, leading to the unusual situation where an organelle genome is far outnumbered by its nuclear counterparts. As a result, comparative mitogenome studies are in their infancy and some key aspects of genome evolution are still known mainly from pre-genome, qualitative methods. To help address these limitations, we combined machine learning and in silico enrichment of mitochondrial-like long reads to assemble the bacterial-sized mitogenome of Norway spruce (Pinaceae: Picea abies). We conducted comparative analyses of repeat abundance, intergenomic transfers, substitution and rearrangement rates, and estimated repeat-by-repeat homologous recombination rates. Prompted by our discovery of highly recombinogenic small repeats in P. abies, we assessed the genomic support for the prevailing hypothesis that intramolecular recombination is predominantly driven by repeat length, with larger repeats facilitating DNA exchange more readily. Overall, we found mixed support for this view: recombination dynamics were heterogeneous across vascular plants and highly active small repeats (ca. 200 bp) were present in about a third of studied mitogenomes. As in previous studies, we did not observe any robust relationships among commonly-studied genome attributes, but we identify variation in recombination rates as a underinvestigated source of plant mitogenome diversity.

genomics↗

bioRxiv2019

Constructing a high-density linkage map to infer the genomic landscape of recombination rate variation in European Aspen (Populus tremula)

The rate of meiotic recombination is one of the central factors determining levels of linkage disequilibrium and the efficiency of natural selection, and many organisms show a positive correlation between local rates of recombination and levels of nucleotide diversity indicating that linked selection is an important factor determining genome-wide levels of nucleotide diversity. Several methods for estimating recombination rates from segregating polymorphisms in natural populations have recently been developed. These methods have been extensively used in part because they are relatively simple to implement even in many non-model organisms, but also because they potentially offer higher resolution than traditional map-based methods. However, thorough comparisons of LD and map-based estimates of recombination are not readily available in plants. Here we present a new, high-resolution linkage map for Populus tremula and use this to estimate variation in recombination rates across the P. tremula genome. We compare these results to recombination rates estimated based on linkage disequilibrium in a large number of unrelated individuals. We also assess how variation in recombination rates is associated with genomic features, such as gene density, repeat density and methylation levels. We find that recombination rates obtained from the two methods largely agree, although the LD-based method identify a number of genomic regions with very high recombination rates that the map-based method fail to detect. Linkage map and LD-based estimates of recombination rates are positively correlated and show similar correlations with other genomic features, showing that both methods can accurately infer recombination rate variation across the genome.

evolutionary biology↗

bioRxiv2018

Seidr: A toolkit for calculation of crowd networks

Gene regulatory and gene co-expression networks are powerful research tools for identifying biological signal within high-dimensional gene expression data. In recent years, research has focused on addressing shortcomings of these techniques with regard to the low signal-to-noise ratio, non-linear interactions and dataset dependent biases of published methods. Furthermore, it has been shown that aggregating networks from multiple methods provides improved results. Despite this, few usable and scalable software tools have been implemented to perform such best-practice analyses. Here, we present Seidr (stylized Seithr), a software toolkit designed to assist scientists in gene regulatory and gene co-expression network inference. Seidr creates community networks to reduce algorithmic bias and utilizes noise corrected network backboning to prune noisy edges in the networks. Using benchmarks in real-world conditions across three eukaryotic model organisms, Saccharomyces cerevisiae, Drosophila melanogaster, and Arabidopsis thaliana, we show that individual algorithms are biased toward functional evidence for certain gene-gene interactions. We further demonstrate that the community network is less biased, providing robust performance across different standards and comparisons for the model organisms. Finally, we apply Seidr to a network of drought stress in Norway spruce (Picea abies (L.) H. Krast) as an example application in a non-model species. We demonstrate the use of a network inferred using Seidr for identifying key components, communities and suggesting gene function for non-annotated genes.

bioinformatics↗

bioRxiv2016

Interspecific plastome recombination reveals ancient reticulate evolution in Picea (Pinaceae)

Plastid sequences are a cornerstone in plant systematic studies and key aspects of their evolution, such as uniparental inheritance and absent recombination, are often treated as axioms. While exceptions to these assumptions can profoundly influence evolutionary inference, detecting them can require extensive sampling, abundant sequence data, and detailed testing. Using advancements in high-throughput sequencing, we analyzed the whole plastomes of 65 accessions of Picea, a genus of ~35 coniferous forest tree species, to test for deviations from canonical plastome evolution. Using complementary hypothesis and data-driven tests, we found evidence for chimeric plastomes generated by interspecific hybridization and recombination in the clade comprising Norway spruce (P. abies) and ten other species. Support for interspecific recombination remained after controlling for sequence saturation, positive selection, and potential alignment artifacts. These results reconcile previous conflicting plastid-based phylogenies and strengthen the mounting evidence of reticulate evolution in Picea. Given the relatively high frequency of hybridization and biparental plastid inheritance in plants, we suggest interspecific plastome recombination may be more widespread than currently appreciated and could underlie reported cases of discordant plastid phylogenies.

genomics↗