Biology subjects

Hawkins, J. S.

Publications and source records attributed to Hawkins, J. S..

8 recordsLinked to original sources

bioRxiv2024

Comprehensive double-mutant analysis of the Bacillus subtilis envelope using double-CRISPRi

Understanding bacterial gene function remains a major biological challenge. Double-mutant genetic interaction (GI) analysis addresses this challenge by uncovering the functional partners of targeted genes, allowing us to associate genes of unknown function with novel pathways and unravel connections between well-studied pathways, but is difficult to implement at the genome-scale. Here, we develop and use double-CRISPRi to systematically quantify genetic interactions at scale in the Bacillus subtilis envelope, including essential genes. We discover > 1000 known and novel genetic interactions. Our analysis pipeline and experimental follow-ups reveal the distinct roles of paralogous genes such as the mreB and mbl actin homologs, and identify new genes involved in the well-studied process of cell division. Overall, our study provides valuable insights into gene function and demonstrates the utility of double-CRISPRi for high-throughput dissection of bacterial gene networks, providing a blueprint for future studies in diverse bacterial species.

genomics↗

bioRxiv2020

QTL mapping in an interspecific sorghum population uncovers plasma-membrane intrinsic proteins as key regulators of salinity tolerance

Salt stress impedes plant growth and disrupts normal metabolic processes, resulting in decreased biomass and increased leaf senescence. Therefore, the ability of a plant to maintain biomass when exposed to salinity stress is critical for the production of salt tolerant crops. To identify the genetic basis of salt tolerance in an agronomically important grain crop, we used a recombinant inbred line (RIL) population derived from an interspecific cross between domesticated Sorghum bicolor (inbred Tx7000) and a wild relative, Sorghum propinquum. A high-density genetic map was generated from 177 F3:5 RILs and covered the 10 Sorghum chromosomes with 1991 markers. The genetic map was used to identify 19 total QTL related to plant growth and overall health in optimal and saline conditions. Of these 19 QTL detected, 10 were specific to the salt stress response. The salt-responsive QTL contained numerous genes that have been previously shown to play a role in ionic tolerance, tissue tolerance, and osmotic tolerance, including many aquaporins.

genomics↗

bioRxiv2020

Integration of high-density genetic mapping with transcriptome analysis uncovers numerous agronomic QTL and reveals candidate genes for the control of tillering in sorghum

Phenotypes such as branching, photoperiod sensitivity, and height were modified during plant domestication and crop improvement. Here, we perform quantitative trait locus (QTL) mapping of these and other agronomic traits in a recombinant inbred line (RIL) population derived from an interspecific cross between Sorghum propinquum and Sorghum bicolor inbred Tx7000. Using low-coverage Illumina sequencing and a bin-mapping approach, we generated ~1920 bin markers spanning ~875 cM. Phenotyping data were collected and analyzed from two field locations and one greenhouse experiment for six agronomic traits, thereby identifying a total of 30 QTL. Many of these QTL were penetrant across environments and co-mapped with major QTL identified in other studies. Other QTL uncovered new genomic regions associated with these traits, and some of these were environment-specific in their action. To further dissect the genetic underpinnings of tillering, we complemented QTL analysis with transcriptomics, identifying 6189 genes that were differentially expressed during tiller bud elongation. We identified genes such as Dormancy Associated Protein 1 (DRM1) in addition to various transcription factors that are differentially expressed in comparisons of dormant to elongating tiller buds and lie within tillering QTL, suggesting that these genes are key regulators of tiller elongation in sorghum. Our study demonstrates the usefulness of this RIL population in detecting domestication and improvement-associated genes in sorghum, thus providing a valuable resource for genetic investigation and improvement to the sorghum community.Competing Interest StatementThe authors have declared no competing interest.View Full Text

genetics↗

bioRxiv2020

Evolutionary dynamics of transposable elements following a shared polyploidization event in the tribe Andropogoneae

Both polyploidization and transposable element (TE) activity are known to be major drivers of plant genome evolution. Here, we utilize the Zea-Tripsacum clade to investigate TE activity and accumulation after a recent shared polyploidization event. Comparisons of TE evolutionary dynamics in various Zea and Tripsacum species, in addition to two closely related diploid species, Urelytrum digitatum and Sorghum bicolor, revealed existing variation in repeat content among all taxa included in the study. The repeat composition of Urelytrum is more similar to that of Zea and Tripsacum compared to Sorghum, despite the similarity in genome size with the latter. Although the genomes of all species studied had abundant LTR-retrotransposons, we observed an expansion of the copia superfamily, specifically in Z. mays and T. dactyloides, species that have adapted to more temperate environments. Additional analyses of the genomic distribution of these copia elements provided evidence of biased insertions near genes involved in various biological processes including plant development, defense, and macromolecule biosynthesis. The lack of copia insertions near the orthologous genes in S. bicolor suggests that duplicate gene copies generated during polyploidization may offer novel neutral sites for TEs to insert, thereby providing an avenue for subfunctionalization via TE insertional mutagenesis.

evolutionary biology↗

bioRxiv2019

Phenotypic and physiological responses to salt exposure in Sorghum reveal diversity among domesticated landraces

Soil salinity negatively impacts plant function, development, and yield. Sorghum bicolor is a staple crop known to be drought tolerant, to have adapted to a variety of conditions, and to contain significant standing genetic diversity, making it an exemplary species to study phenotypic and physiological variation in salinity tolerance. In our study, a diverse group of sorghum landraces and accessions was first rank-ordered for salinity tolerance and then individuals spanning a wide range of response were analyzed for foliar proline and ion accumulation. We found that, while proline is often a good indicator of osmotic adjustment and is historically associated with increased salt tolerance, proline accumulation in sorghum reflects stress-response injury rather than acclimation. When combining ion profiles with growth responses and stress tolerance indices, the variation observed in tolerance was similarly not a sole result of Na+ accumulation, but rather reflected accession-specific mechanisms that may integrate these and other metabolic responses. When we compared variation in tolerance to phylogenetic relationships, we conclude that the most parsimonious explanation for the variation observed among accessions is that salinity tolerance was acquired early during domestication and was subsequently maintained or lost in diverged lineages during improvement in areas that vary in soil salinity.

plant biology↗

bioRxiv2019

Modulated efficacy CRISPRi reveals evolutionary conservation of essential gene expression-fitness relationships in bacteria

Essential genes are the central hubs of cellular networks. Despite their importance, the lack of high-throughput methods for titrating their expression has limited our understanding of the fitness landscapes against which essential gene expression levels are optimized. We developed a modified CRISPRi system leveraging the predictable reduction in efficacy of imperfectly matched sgRNAs to generate specific levels of CRISPRi activity and demonstrate its broad applicability in bacteria. Using libraries of mismatched sgRNAs, we characterized the expression-fitness relationships of essential genes in Escherichia coli and Bacillus subtilis. Remarkably, these relationships co-vary by pathway and are predominantly conserved between E. coli and B. subtilis despite ~ 2 billion years of evolutionary separation, suggesting that deeply conserved tradeoffs underlie bacterial homeostasis.\n\nOne Sentence SummaryBacterial essential genes have varying responses to CRISPRi knockdown that are largely conserved across ~2 billion years of evolution.

systems biology↗

bioRxiv2019

Titrating gene expression with series of systematically compromised CRISPR guide RNAs

Biological phenotypes arise from the degrees to which genes are expressed, but the lack of tools to precisely control gene expression limits our ability to evaluate the underlying expression-phenotype relationships. Here, we describe a readily implementable approach to titrate expression of human genes using series of systematically compromised sgRNAs and CRISPR interference. We empirically characterize the activities of compromised sgRNAs using large-scale measurements across multiple cell models and derive the rules governing sgRNA activity using deep learning, enabling construction of a compact sgRNA library to titrate expression of [~]2,400 genes involved in central cell biology and a genome-wide in silico library. Staging cells along a continuum of gene expression levels combined with rich single-cell RNA-seq readout reveals gene-specific expression-phenotype relationships with expression level-specific responses. Our work provides a general tool to control gene expression, with applications ranging from tuning biochemical pathways to identifying suppressors for diseases of dysregulated gene expression.

genomics↗

bioRxiv2018

Mobile-CRISPRi: Enabling Genetic Analysis of Diverse Bacteria

Introductory paragraphThe vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. CRISPRi, a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs (sgRNAs), has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to human. However, the difficulty of establishing effective CRISPRi systems in non-model bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish \"Mobile-CRISPRi\", a suite of CRISPRi systems that combine modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in Proteobacteria and Firmicutes at the individual gene scale by examining drug-gene synergies and at the library scale by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microbe interactions.

microbiology↗