Biology subjects

Dubinett, S.

Publications and source records attributed to Dubinett, S..

3 recordsLinked to original sources

bioRxiv2026

FORGE-CRISPR: Reusable Modules for Focused CRISPR Library Construction

Pooled CRISPR screening has become a widely used approach for functional genomics, yet the construction of screening libraries remains tightly coupled to specific vector architectures and experimental formats. Incorporation of barcodes, multiplexed guide configurations, alternative fluorophores, selectable markers, or distinct screening modalities often requires reconstruction of entire libraries, even when the underlying biological content remains unchanged. These limitations are particularly acute in engineered cellular systems that already contain reporters, knock-in alleles, or pre-existing selection markers. Here, we describe FORGE-CRISPR (Functional Oncology Research Genetic Engineering - CRISPR), a CRISPR library-construction system that separates biological guide content from screening-vector context. CRISPR knockout and CRISPR interference guide collections are first converted into reusable guide modules. Barcode modules and second-guide modules are generated separately and combined with guide modules during downstream assembly into screening acceptor vectors. This design allows guide-only, barcoded, and multiplexed libraries to be generated from shared physical components. To support this framework, we developed a manufacturing workflow in which synthetic oligonucleotide pools are converted into reusable FORGE module libraries through PCR amplification, Golden Gate cloning, and background suppression. Using this approach, we constructed nine reusable guide-module libraries ranging from 45 to 4,830 guides (11,303 guides in total), and evaluated library quality through PCR-NGS analysis of guide representation and abundance distributions. As content for these libraries, we defined a modular, non-overlapping set of focused target collections, termed the druggable oncology genome, by partitioning druggable targets according to clinical development status and dependency distribution in DepMap, while drawing guide sequences from established, experimentally validated genome-wide libraries. We report four resources: reusable guide, barcode, and second-guide modules; compatible screening acceptor vectors; a manufacturing and PCR-NGS quality-control workflow demonstrated across nine guide-module libraries; and a versioned set of focused druggable-oncology target collections.

Molecular Biology↗

bioRxiv2020

Resolving the spatial and cellular architecture of lung adenocarcinoma by multi-region single-cell sequencing

Little is known of the geospatial architecture of individual cell populations in lung adenocarcinoma (LUAD) evolution. Here, we perform single-cell RNA sequencing of 186,916 cells from 5 early-stage LUADs and 14 multi-region normal lung tissues of defined spatial proximities from the tumors. We show that cellular lineages, states, and transcriptomic features geospatially evolve across normal regions to LUADs. LUADs also exhibit pronounced intratumor cell heterogeneity within single sites and transcriptional lineage-plasticity programs. T regulatory cell phenotypes are increased in normal tissues with proximity to LUAD, in contrast to diminished signatures and fractions of cytotoxic CD8+ T cells, antigen-presenting macrophages and inflammatory dendritic cells. We further find that the LUAD ligand-receptor interactome harbors increased expression of epithelial CD24 which mediates pro-tumor phenotypes. These data provide a spatial atlas of LUAD evolution, and a resource for identification of targets for its treatment. Statement of significanceThe geospatial ecosystem of the peripheral lung and early-stage LUAD is not known. Our multi-region single-cell sequencing analyses unravel cell populations, states, and phenotypes in the spatial and ecological evolution of LUAD from the lung that comprise high-potential targets for early interception.

cancer biology↗

bioRxiv2020

Single-cell analysis of human lung epithelia reveals concomitant expression of the SARS-CoV-2 receptor ACE2 with multiple virus receptors and scavengers in alveolar type II cells

The novel coronavirus SARS-CoV-2 was identified as the causative agent of the ongoing pandemic COVID 19. COVID-19-associated deaths are mainly attributed to severe pneumonia and respiratory failure. Recent work demonstrated that SARS-CoV-2 binds to angiotensin converting enzyme 2 (ACE2) in the lung. To better understand ACE2 abundance and expression patterns in the lung we interrogated our in-house single-cell RNA-sequencing dataset containing 70,085 EPCAM+ lung epithelial cells from paired normal and lung adenocarcinoma tissues. Transcriptomic analysis revealed a diverse repertoire of airway lineages that included alveolar type I and II, bronchioalveolar, club/secretory, quiescent and proliferating basal, ciliated and malignant cells as well as rare populations such as ionocytes. While the fraction of lung epithelial cells expressing ACE2 was low (1.7% overall), alveolar type II (AT2, 2.2% ACE2+) cells exhibited highest levels of ACE2 expression among all cell subsets. Further analysis of the AT2 compartment (n = 27,235 cells) revealed a number of genes co-expressed with ACE2 that are important for lung pathobiology including those associated with chronic obstructive pulmonary disease (COPD; HHIP), pneumonia and infection (FGG and C4BPA) as well as malarial/bacterial (CD36) and viral (DMBT1) scavenging which, for the most part, were increased in smoker versus light or non-smoker cells. Notably, DMBT1 was highly expressed in AT2 cells relative to other lung epithelial subsets and its expression positively correlated with ACE2. We describe a population of ACE2-positive AT2 cells that co-express pathogen (including viral) receptors (e.g. DMBT1) with crucial roles in host defense thus comprising plausible phenotypic targets for treatment of COVID-19.

genomics↗