Biology subjects

Guell, M.

Publications and source records attributed to Guell, M..

7 recordsLinked to original sources

bioRxiv2025

Genome editing of phylogenetically distinct bacteria using portable retron-mediated recombineering

Advanced genome editing technologies have enabled rapid and flexible rewriting of the Escherichia coli genome, benefiting fundamental biology and biomanufacturing. Unfortunately, some of the most useful technologies to advance genome editing in E. coli have not yet been ported into other bacterial species. For instance, the addition of bacterial retrons to the genome editing toolbox has increased the efficiency of recombineering in E. coli by enabling sustained, abundant production of ssDNA recombineering donors by reverse transcription that install flexible, precise edits in the prokaryotic chromosome. To extend the utility of this technology beyond E. coli, we surveyed the portability and versatility of retron-mediated recombineering across three different bacterial phyla (Proteobacteria, Bacillota and Actinomycetota) and a total of 15 different species. We found that retron recombineering is functional in all species tested, reaching editing efficiencies above 20% in six of them, above 40% in three of them, and above 90% in two of them. We also tested the extension of the recombitron architecture optimizations and strain backgrounds in a subset of hosts to additionally increase editing rates. The broad recombitron survey carried out in this study forms the basis for widespread use of retron-derived technologies through the whole Bacteria domain.

bioengineering↗

bioRxiv2022

New insights into the role of Cutibacterium acnes-derived extracellular vesicles in inflammatory skin disorders

BackgroundCutibacterium acnes (C. acnes) is one of the most prevalent bacteria that form the human skin microbiota and, depending on multifactorial conditions it can help to maintain the skin homeostasis. Actually, different phylotypes of C. acnes have been associated with different degrees of acne vulgaris development, while others, such as the H1 subtype, have been detected in patients with non-acneic skin. However, due to the physiology of the skin, the skin microbiota neither has direct access to the skins sebaceous glands nor to the main immune cells, as they are protected by a sebum layer. Therefore, the inter-kingdom communication relies on secreted factors and bacterial extracellular vesicles (EVs). In this context, the purpose of this project was to study the role of EVs secreted by three different phylotypes of C. acnes (A1 as pathogenic, H1 as beneficial and H2 as commensal). ResultsMain findings showed that the proteomic profile of the cargo embodied in the EVs reflects unique characteristics of the different C. acnes phylotypes in terms of lifestyle, survival and virulence. Moreover, in vitro skin models showed an extended pro-inflammatory modulation of A1 EVs, while H1 EVs displayed a high sebum-reducing potential. ConclusionsThis study has highlighted the role of C. acnes EVs as key modulators during skin alterations, specially H1 EVs as an alternative based-natural treatment to fight acne vulgaris symptomatology.

immunology↗

bioRxiv2022

Synergic homology directed recombination by PRDM9 meiotic factor

Genome editing requires precision to broadly move on to industrial and clinical applications. For this reason, homologous directed repair (HDR) is one of the preferred methods for small edits, other than knock-outs. However, HDR has low efficiency. Current investigations to enhance HDR have mainly gone in the direction of finding non-homologous end joining (NHEJ) inhibitors. NHEJ is crucial for cellular integrity, then the inhibition of this pathway is detrimental for the correct survival of living entities. In other studies, a second opportunity is given to HDR by targeting the byproducts of NHEJ, using an extra gRNA. In this study, we propose the use of a meiotic factor, PRDM9, to directly enhance homology recombination. Through the exploration of combinatorial factors and donor design, we have established an optimized protocol for HDR. PRDM9-Cas9 fusion combined with CtIP improves HDR/NHEJ ratio. In addition, we have validated this combinatorial approach for small edits through a traffic light reporter system, as well as for longer edits with a split-GFP reporter system.

synthetic biology↗

bioRxiv2022

INSERT-seq enables high resolution mapping of genomically integrated DNA using nanopore sequencing

Comprehensive characterization of genome engineering with viral vectors, transposons, CRISPR/Cas mediated DNA integration and other DNA editors remains relevant for their development and safe use in human gene therapy. Currently, described methods for measuring DNA integration in edited cells rely on short read based technologies. Due to the repetitive nature of the human genome, short read based methods can potentially overlook insertion events in repetitive regions. We modelled the impact of read length in resolving insertion sites, which suggested a significant drop in insertion site detection with shorter read length. Based on that, we developed a method that combines targeted amplification of integrated DNA, UMI-based correction of PCR bias and Oxford Nanopore long-read sequencing for robust analysis of DNA integration in a genome. This method, called INSERT-seq, is capable of detecting events occurring at a frequency of up to 0.1%. INSERT-seq presents a complete handling of all insertions independently of repeat size. The experimental pipeline improves the number mappable insertions at repetitive regions by 7.3% and repeats larger than the long read sequencing size are processed computationally to perform a peak calling in a repeat database. INSERT-seq is a simple, cheap and robust method to quantitatively characterise DNA integration in diverse ex-vivo and in-vivo samples.

genomics↗

bioRxiv2020

CRISPR-mediated biocontainment

We have exploited the repetitive nature of transposable elements of the human genome to generate synthetic circuits. Transposable elements such as LINE-1 and Alu have successfully replicated in mammalian genomes throughout evolution to reach a copy number ranging from thousands to more than a million. Targeting these repetitive elements with programmable DNA nucleases such as CRISPR-Cas9 rapidly induce extremely high levels of cell death. We use this genotoxic feature to build synthetic biocontainment circuits: CRISPR defense system (CRISPR-DS) capable of preventing CRISPR genome editing, and we introduce the proof-of-concept of CRISPR Safety-Switch, an inducible, stringent and non-leaky kill-switch capable of clearing out cell lines resistant to DNA breaks.

synthetic biology↗

bioRxiv2019

Extensive Mammalian Germline Genome Engineering

Xenotransplantation, specifically the use of porcine organs for human transplantation, has long been sought after as an alternative for patients suffering from organ failure. However, clinical application of this approach has been impeded by two main hurdles: 1) risk of transmission of porcine endogenous retroviruses (PERVs) and 2) molecular incompatibilities between donor pigs and humans which culminate in rejection of the graft. We previously demonstrated that all 25 copies of the PERV elements in the pig genome could be inactivated and live pigs successfully generated. In this study, we improved the scale of porcine germline editing from targeting a single repetitive locus with CRISPR to engineering 13 different genes using multiple genome engineering methods. we engineered the pig genome at 42 alleles using CRISPR-Cas9 and transposon and produced PERVKO{middle dot}3KO{middle dot}9TG pigs which carry PERV inactivation, xeno-antigen KO and 9 effective human transgenes. The engineered pigs exhibit normal physiology, fertility, and germline transmission of the edited alleles. In vitro assays demonstrated that these pigs gain significant resistance to human humoral and cell mediated damage, and coagulation dysregulations, similar to that of allotransplantation. Successful creation of PERVKO{middle dot}3KO{middle dot}9TG pigs represents a significant step forward towards safe and effective porcine xenotransplantation, which also represents a synthetic biology accomplishment of engineering novel functions in a living organism. One Sentence SummaryExtensive genome engineering is applied to modify pigs for safe and immune compatible organs for human transplantation

synthetic biology↗

bioRxiv2018

Skin microbiome modulation induced by probiotic solutions

BackgroundThe skin is colonized by a large number of microorganisms, of which most are beneficial or harmless. However, disease states of skin have specific microbiome compositions that are different from those of healthy skin. Gut microbiome modulation through fecal transplant has proven as a valid therapeutic strategy in diseases such as Clostridium difficile infections. Therefore, techniques to modulate the skin microbiome composition may become an interesting therapeutic option in diseases affecting the skin such as psoriasis or acnes vulgaris.\n\nHere we have used mixtures of different skin microbiome components to alter the composition of a recipient skin microbiome.\n\nResultsWe show that after sequential applications of a donor microbiome, the recipient microbiome becomes similar to that of the donor. After intervention, an initial, week-long phase is characterized by dominance of donor strains. The level of engraftment depends on the composition of the recipient and donor microbiomes, and the applied bacterial load. We observed higher engraftment using a multi-strain donor solution with recipient skin rich in Cutibacterium acnes subtype H1 and Leifsonia.\n\nConclusionsWe have demonstrated the use of living bacteria to modulate skin microbiome composition.

microbiology↗