Biology subjects

Harland-Dunaway, M.

Publications and source records attributed to Harland-Dunaway, M..

2 recordsLinked to original sources

bioRxiv2024

Optimized genome-wide CRISPR screening enables rapid engineering of growth-based phenotypes in Yarrowia lipolytica

CRISPR-Cas9 functional genomic screens uncover gene targets linked to various phenotypes for metabolic engineering with remarkable efficiency. However, these genome-wide screens face a number of design challenges, including variable guide RNA activity, ensuring sufficient genome coverage, and maintaining high transformation efficiencies to ensure full library representation. These challenges are prevalent in non-conventional yeast, many of which exhibit traits that are well suited to metabolic engineering and bioprocessing. To address these hurdles in the oleaginous yeast Yarrowia lipolytica, we designed a compact, high-activity genome-wide sgRNA library. The library was designed using DeepGuide, a sgRNA activity prediction algorithm, and a large dataset of [~]50,000 sgRNAs with known activity. Three guides per gene enables redundant targeting of 98.8% of genes in the genome in a library of 23,900 sgRNAs. We deployed the optimized library to uncover genes essential to the tolerance of acetate, a promising alternative carbon source, and various hydrocarbons present in many waste streams. Our screens yielded several gene knockouts that improve acetate tolerance on their own and as double knockouts in media containing acetate as the sole carbon source. Analysis of the hydrocarbon screens revealed genes related to fatty acid and alkane metabolism in Y. lipolytica. The optimized CRISPR gRNA library and its successful use in Y. lipolytica led to the discovery of alternative carbon source-related genes and provides a workflow for creating high-activity, compact genome-wide libraries for strain engineering. HighlightsO_LIDesigned a compact, high activity CRISPR sgRNA knockout library for Yarrowia lipolytica. C_LIO_LIDeveloped an efficient pipeline for discovering genes involved in alternative carbon-source utilization. C_LIO_LIIdentified single and double gene knockouts that improve growth on acetate. C_LIO_LIIdentified genes with improved fitness and essentiality for hydrocarbon growth. C_LI

synthetic biology↗

bioRxiv2018

Extreme nuclear branching in healthy epidermal cells of the Xenopus tail fin

Changes in nuclear morphology contribute to regulation of complex cell properties, including differentiation and tissue elasticity. Perturbations of nuclear morphology are associated with pathologies that include, progeria, cancer, and muscular dystrophy. The mechanisms governing nuclear shape changes in healthy cells remain poorly understood, partially because there are few healthy models of nuclear shape variation. Here, we introduce nuclear branching in epidermal fin cells of Xenopus tropicalis as a model for extreme variation of nuclear morphology in a diverse population of healthy cells. We find that nuclear branching arises and elaborates during embryonic development. They contain broadly distributed marks of transcriptionally active chromatin and heterochromatin and have active cell cycles. We find that nuclear branches are disrupted by loss of filamentous actin and depend on epidermal expression of the nuclear lamina protein Lamin B1. Inhibition of nuclear branching disrupts fin morphology, suggesting that nuclear branching may be involved in fin development. This study introduces the nuclei of the fin as a powerful new model for extreme nuclear morphology in healthy cells to complement studies of nuclear shape variation in pathological contexts.\n\nList of abbreviations and symbolsLINC-Linker of Nucleoskeleton and Cytoskeleton, HGPS - Hutchinson-Gilford Progeria Syndrome, TEM - Transmission electron microscopy, PH3 - Phosphorylated Histone 3, Lat B - Latrunculin B, WT-Wild type, Cyto D-Cytochalasin D, Lmnb1 CRISPR - lmnb1 mutants generated by CRISPR/Cas9, E-Lmnb1 CRISPR - epidermal specific lmnb1 mutants generated by CRISPR/Cas9, Scrmbl-tadpoles injected with a scrambled version of the lmnb1 targeted sgRNA, Lmnb1-rod - dominant-negative Lamin B1 containing only the rod domain\n\nSummary StatementNuclei are highly branched throughout the heterogeneous population of healthy epidermal cells that comprise the Xenopus tail fin periphery, and disruption of nuclear branching mechanisms results in improper fin morphology.

cell biology↗