Biology subjects

Gibson, D.

Publications and source records attributed to Gibson, D..

6 recordsLinked to original sources

bioRxiv2026

Optimized parameters for Cas9 CRISPR interference library design

CRISPR interference (CRISPRi) is a powerful technology for studying loss-of-function phenotypes, enabling transient and reversible control of gene expression without the introduction of double-stranded DNA breaks. The cost of conducting large-scale CRISPR screens necessitates the selection of effective and specific sgRNAs for the design of compact libraries. While several genome-wide Cas9 CRISPRi libraries have been created, updates to transcript annotations, the generation of higher-resolution chromatin accessibility datasets and the development of newer on-target prediction models motivate an updated CRISPRi library design approach. Here, we generate large CRISPRi datasets tiling essential and nonessential genes. We compare the performance of multiple KRAB domain systems, develop an updated CRISPRi-specific on-target scoring scheme, and quantitatively characterize off-target effects associated with seed sequence patterns. We leverage these findings to design an optimized Cas9 CRISPRi library, Katsano, and validate its performance with genome-wide viability screens.

bioengineering↗

bioRxiv2026

Protein-only centromeric chromatin assembly streamlines human artificial chromosome formation

Human artificial chromosomes (HACs) are inherited through cell divisions alongside natural chromosomes, serving as tools for interrogating chromosomal elements and as vectors for large genetic cargoes (1-5). Despite recent progress (6-8), large (i.e., multiple Mb) HACs have not been reported. Further, HAC formation via epigenetic seeding of centromeric chromatin currently requires prior engineering of recipient human cells (6-8), hampering their potential deployment in many useful cell types. Here, we designed, built, and delivered to human cells a 2 Mb HAC construct that is [~]3 times larger than the prior generation. We also report a robust epigenetic centromere seeding approach that initiates immediately upon delivery to the human cell cytoplasm and bypasses genetic engineering of target cells. The HACs are then faithfully inherited in the absence of selection. Thus, formation of functional centromeric chromatin in the same cell cycle of HAC delivery drives high efficiency HAC formation. TeaserA system for efficient protein-only centromere seeding permits visualizing the first step of HAC formation

synthetic biology↗

bioRxiv2026

Taking genomics outdoors: linking local adaptation, trait variation, and gene expression in grass ecotypes across a rainfall gradient

With increasing droughts, understanding local adaptation and drought tolerance in ecologically dominant species is crucial for enhancing ecosystem resilience. We leveraged a long-term reciprocal garden to assess local adaptation and drought responses in Andropogon gerardi, a foundation grassland species in the US Great Plains. Our objectives were to identify adaptive traits, explore gene expression responses across a rainfall gradient and under experimental drought and integrate trait-based analyses with gene expression profiles to test for local adaptation. Reciprocal gardens, established a decade ago, are composed of different A. gerardi ecotypes sourced along a rainfall gradient (MAP 480-1167mm yr-1) and sown as ecological communities. Rainout shelters imposed experimental drought. We hypothesized that ecotypes should perform best in their homesite, reflecting local adaptation. The dry ecotype should perform best under rainouts and exhibit traits and expression profiles favoring drought tolerance; the wet ecotype should favor traits and gene expression enhancing growth and resource acquisition. We found ecotypes had highest biomass and cover in their homesite, confirming local adaptation. Under experimental drought, the dry ecotype demonstrated improved performance at the wet site, confirming its adaptive value under water limitation. The dry ecotype showed stress-tolerance strategies (shorter, more water-efficient, upregulated drought-response genes), while the wet ecotype emphasized growth strategies (taller, higher biomass, upregulation of growth hormone gibberellin). Using co-expression networks, gene clusters linked adaptive traits, revealing genetic mechanisms of adaptation. Results advance our understanding of adaptation by linking gene expression and trait variation to drought responses in locally-adapted ecotypes, informing ecotype climate-matching under drought.

genomics↗

bioRxiv2025

Synthetic cargo adaptors reveal molecular features that can enhance dynein activation

Cytoplasmic dynein-1 (dynein) facilitates the microtubule-based retrograde trafficking of all cellular cargo. To become active, dynein binds dynactin and one of many cargo-specific adaptors to form the active transport complex. Despite having similar structures, active transport complexes assembled with different adaptors move with different properties in vitro. To explore how adaptors differentially activate dynein, we engineered a library of synthetic adaptors and characterized their ability to activate dynein using in vitro reconstitution and cell-based trafficking assays. We found that the apparent motility of dynein is highly plastic and tunable by the adaptor sequence and that it is possible to engineer adaptors that outperform endogenous adaptors ability to generate highly motile active transport complexes. We also found that different adaptors support distinct trafficking behavior and cargo movement in cells. These findings provide insight into how dynein motility is modulated to meet the unique trafficking requirements of all cellular cargo.

biophysics↗

bioRxiv2024

A Novel Cisplatin-Based Prodrug Inhibits Lysine Deacetylases, Suppresses Nucleotide Excision Repair, and Overcomes Resistance

Cisplatin [cis-diamminedichloroplatinum(II)] is a widely used chemotherapeutic agent that induces cytotoxicity primarily through DNA damage, but drug resistance severely limits its efficacy and use. Cisplatin resistance is complex and multifactorial, involving DNA repair via nucleotide excision repair (NER), and overexpression of lysine deacetylases (KDACs), which reduce chromatin accessibility and alter transcription regulation. The combination of cisplatin and KDAC inhibitors has shown promise in improving treatment efficacy. This improved efficacy has been attributed to increased drug sensitivity due to higher chromatin accessibility, however, this hypothesis has not been validated. In this study, we synthesized a novel Pt(IV) derivative, cct-[Pt(NH3)2Cl2(VPA)(PhB)] (cPVP), which combines cisplatin and two KDAC inhibitors, phenyl butyrate and valproic acid. This triple-action prodrug enabled the simultaneous targeting of multiple cancer-related pathways. Compared to cisplatin, cPVP exhibited significantly enhanced damage formation and cytotoxicity. High-resolution mapping of cisplatin damage and repair, however, does not attribute the enhanced damage sensitivity to chromatin accessibility, but rather to increased drug uptake and inhibition of nucleotide excision repair. Moreover, cPVP treatment increased survival in a mouse mesothelioma model, and prevented the development of resistance to both cisplatin and itself in cancer cells. Our findings shed new light on the effect of KDAC inhibition on cisplatin treatment, and suggest that cPVP could serve as a promising alternative to cisplatin in the clinic.

molecular biology↗

bioRxiv2024

CCSer2 gates dynein activity at the cell periphery

Cytoplasmic dynein-1 (dynein) is a microtubule-associated, minus end-directed motor that traffics hundreds of different cargos. Dynein must discriminate between cargos and traffic them at the appropriate time from the correct cellular region. How dyneins trafficking activity is regulated in time or cellular space remains poorly understood. Here, we identify CCSer2 as the first known protein to gate dynein activity in the spatial dimension. CCSer2 promotes the migration of developing zebrafish primordium cells and of cultured human cells by facilitating the trafficking of cargos that are acted on by cortically localized dynein. CCSer2 inhibits the interaction between dynein and its regulator Ndel1 exclusively at the cell periphery, resulting in localized dynein activation. Our findings suggest that the spatial specificity of dynein is achieved by the localization of proteins that disinhibit Ndel1. We propose that CCSer2 defines a broader class of proteins that activate dynein in distinct microenvironments via Ndel1 inhibition.

cell biology↗