Biology subjects

De Marco, F.

Publications and source records attributed to De Marco, F..

4 recordsLinked to original sources

bioRxiv2025

Overcoming lentiviral delivery limitations in hard-to-transduce suspension cells for genome-wide CRISPR screening

Lentiviral vectors are a cornerstone delivery modality of biomedical research, renowned for their ability to stably integrate genetic material into the host genome, enabling sustained transgene expression and long-term genetic manipulation. These properties make them indispensable tools in functional genomics and genome engineering, particularly for delivering molecular components in high-throughput CRISPR screening, a powerful approach for uncovering the genetic basis of complex cellular mechanisms and phenotypes. However, challenges such as lentiviral-induced recombination, unpredictable integration profiles, and variable susceptibility of target cells to transduction can introduce noise and compromise experimental outcomes. In this study, we selected two suspension-adapted mammalian cell lines, Chinese Hamster Ovary cells CHO-K1 and Human Embryonic Kidney cells HEK293-6E, due to their widespread use in recombinant protein production. Recognizing the influence of intrinsic cell line properties and transduction methodology, we compared two distinct procedures: spinoculation and static transduction. By implementing a two-step static transduction protocol, we achieved significantly higher transduction efficiencies while minimizing cellular stress, streamlining workflows, and eliminating scalability limitations inherent to large-scale lentiviral applications like genome-wide CRISPR screens. To further characterize the variation in lentiviral integration, we used droplet digital PCR (ddPCR) to quantify copy number variation (CNV) both at the pooled population level and within individual clonal isolates. This comprehensive analysis underscores the robustness of our optimized protocol in enhancing transduction efficiency in difficult-to-transduce suspension cell lines. It further emphasizes the importance of carefully modulating infection rates to limit multiple integrations, ensuring the accuracy and consistency required for large-scale functional genomics applications.

molecular biology↗

bioRxiv2025

A genome-scale CRISPR deletion screen in Chinese Hamster Ovary cells reveals essential regions of the coding and non-coding genome

The biopharmaceutical sector relies on CHO cells to investigate biological processes and as the preferred host for production of biotherapeutics. Simultaneously, advancements in CHO cell genome assembly have provided insights for developing sophisticated genetic engineering strategies. While the majority of these efforts have focused on coding genes, with some interest in transcribed non-coding RNAs (e.g., microRNAs and lncRNAs), there remains a lack of genome-wide systematic studies that precisely examine the remaining 90% of the genome. This unannotated "dark matter" includes regulatory elements and other, poorly understood or characterized functionality of the genome that may be potentially critical for cell survival. In this study, we deployed a genome-scale CRISPR screening platform with 112,272 paired guide RNAs targeting 14,034 genomic regions for complete deletion of 150 kb long sections. This platform enabled the execution of a negative screen that selectively identified dying cells to determine regions essential for cell survival. By using paired gRNAs, we overcame the intrinsic limitations of traditional frameshift strategies, which will likely have little or no effect on the non-coding genome. This study revealed 427 regions essential for CHO survival, many of which currently lack gene annotation or known function. For these regions we present annotation status, transcriptional activity as well as annotated chromatin states such as enhancers. Selected regions, specifically those that were negative for all the above, were individually deleted for confirmation. This work sheds a novel light on a substantial part of the mammalian genome which is traditionally difficult to investigate and therefore, neglected.

cell biology↗

bioRxiv2020

Role of the SUT1 and SUT2 sugar transporters during stolbur phytoplasma infection in tomato

Phytoplasmas inhabit phloem sieve elements and cause abnormal growth and altered sugar partitioning. But how they interact with phloem functions is not clearly known. The phloem responses were investigated in tomato infected by Candidatus Phytoplasma solani, at the beginning of the symptomatic stage of infection, both in symptomatic and asymptomatic leaves, the first symptoms appearing in the sink top leaf at the stem apex. Antisense lines impaired in the phloem sucrose transporters SUT1 and SUT2 were included. The infection in source leaves was not associated with symptoms. In the symptomatic, sink leaf, yellowing and leaf curling was associated with higher starch accumulation and expression of defense genes. The transcriptional analysis of symptomatic leaf midribs indicated that transcript levels for genes acting in the glycolysis and peroxisome metabolism in infected plants differed from these in non-infected plants. Phytoplasma multiplied actively in at least three additional lower leaves although they were symptomless, with no sign of activation of defense markers, although the rate of exudation of sucrose from these symptomless, source leaves was lower for infected plants. A few metabolites in phloem-enriched exudate were affected by the infection, such as glycolate and aspartate, and some of them were also affected in the control SUT1- and SUT2- antisense lines, in which sucrose retrieval or release in the sieve elements are impaired. A metabolic switch could explain the delivery of more glycolate into the sieve elements of infected plants. The findings suggest a link between sugar transport and redox homeostasis. One sentence summaryAn impairment of sucrose retrieval and release in the sieve elements occurs during phytoplasma infection, associated with changes in sugar and peroxisome metabolism

plant biology↗

bioRxiv2018

Synchrotron FTIR and Raman spectroscopy provide unique spectral fingerprints for Arabidopsis floral stem vascular tissues

Cell walls are highly complex structures that are modified during plant growth and development. For example, the development of phloem and xylem vascular cells, which participate in the transport of sugars and water as well as support, can be influenced by cell-specific cell wall composition. Here, we used synchrotron radiation-based infrared (SR-FTIR) and Raman spectroscopy to analyze the cell wall composition of wild-type and double mutant sweet11-1sweet12-1, which impairs sugar transport, Arabidopsis floral stem vascular tissue. The SR-FTIR spectra showed that in addition to modified xylem cell wall composition, phloem cell walls in the double mutant line were characterized by modified hemicellulose composition. Moreover, combining Raman spectroscopy with a Classification and Regression Tree (CART) method identified combinations of Raman shifts that could distinguish xylem vessels and fibers. Additionally, the disruption of SWEET11 and SWEET12 genes impacts xylem cell wall composition in a cell-specific manner, with changes in hemicelluloses and cellulose observed at the xylem vessel interface. These results suggest that the facilitated transport of sugars by transporters that exist between vascular parenchyma cells and conducting cells is important to ensuring correct phloem and xylem cell wall composition.\n\nHighlightCombining vibrational spectroscopy techniques and multivariate analysis shows that the disruption of SWEET genes impacts phloem cell wall composition and that the effect on xylem cell wall composition is cell-specific.

plant biology↗