Biology subjects

Francis, R.

Publications and source records attributed to Francis, R..

10 recordsLinked to original sources

bioRxiv2025

Reinforced CRISPR interference enables reliable multiplex gene repression in phylogenetically distant bacteria

Genetic screens are essential for uncovering novel molecular mechanisms and identifying the functions of hypothetical proteins. CRISPR interference (CRISPRi) is a powerful, programmable, and sequence-specific gene repression technology that can be used for high-throughput screening and targeted gene repression. Despite its ease of use, the initial development of CRISPRi systems is labor-intensive in many non-model organisms. Our goal is to simplify this by establishing a host-agnostic CRISPRi platform that utilizes the serine recombinase-assisted genome engineering (SAGE) system. This system integrates CRISPRi machinery directly into the bacterial chromosome, overcoming the limitations of plasmid-based systems and enabling wide sharing across diverse bacteria. We demonstrate the design and optimization of multiplexed CRISPRi to repress multiple genes simultaneously in phylogenetically distant bacteria. We use a Francisella novicida-derived Cas12a system that processes multiple distinct CRISPR RNAs, each targeting a unique gene sequence, from a single transcript. This allows easy multi-gene repression. By reinforcing gene repression with multiple guides targeting a single gene, we achieve robust genetic perturbations without the need to pre-screen the efficacy of guide RNAs. Using this toolkit, we perturb multiple combinations of growth and visual phenotypes in Pseudomonas fluorescens and demonstrate simultaneous repression of multiple fluorescent proteins to near background levels in bacteria from various other genera. While the tools are directly portable to all SAGE-compatible microbes, we illustrate the utility of SAGE by optimizing CRISPRi performance in Rhodococcus jostii through a combinatorial screen of Cas protein and CRISPR array expression variants. The efficient integration of CRISPRi machinery via the SAGE system paves the way for versatile genetic screening, enabling profound insights into gene functions both in laboratory conditions and relevant naturalistic scenarios.

microbiology↗

bioRxiv2024

Advancing the Application of pXRF for Biological Samples

Point 1: Portable x-ray fluorescent (pXRF) technology provides significant opportunities for rapid, non-destructive data collection in a range of fields of study. However, there are sources of variation and sample assumptions that may influence the data obtained, particularly in biological samples. Point 2: We used representative species for four taxa (fish, mammals, birds, reptiles) to test the precision of replicate scans, and the impact of sample thickness, sample state, scan location and scan time on data obtained from a pXRF. Point 3: We detected significant differences in concentration data due to sample state, scanning time and scanning location for all taxa. Infinite thickness assumptions were met for fish, reptile and mammal representatives at all body locations when samples were thawed, but not dried. Infinite thickness was not met for feathers. Scan time results found in most cases the 40, 60 and 80 second beam times were equivalent. Concentration data across replicate scans were highly correlated. Point 4: The opportunities for the use of pXRF in biological studies are wide-ranging. These findings highlight the considerations required when scanning biological samples to ensure the required data are suitably collected, while maintaining minimal radiation exposure to live animals.

biochemistry↗

bioRxiv2023

Homologous Pairs of Low and High Temperature Originating Proteins Spanning the Known Prokaryotic Universe

Stability of proteins at high temperature has been a topic of interest for many years, as this attribute is favourable for applications ranging from therapeutics to industrial chemical manufacturing. Our current understanding and methods for designing high-temperature stability into target proteins are inadequate. To drive innovation in this space, we have curated a large dataset, learn2thermDB, of protein-temperature examples, totalling 24 million instances, and paired proteins across temperatures based on homology, yielding 69 million protein pairs - orders of magnitude larger than the current largest. This important step of pairing allows for study of high-temperature stability in a sequence-dependent manner in the big data era. The data pipeline is parameterized and open, allowing it to be tuned by downstream users. We further show that the data contains signal for deep learning. This data offers a new doorway towards thermal stability design models.

bioinformatics↗

bioRxiv2022

Kupffer cells dictate hepatic responses to the atherogenic dyslipidemic insult

Apolipoprotein-B (APOB) containing lipoproteins are causative for atherosclerotic cardiovascular disease. Whether the vasculature is the initial responding site or if atherogenic-dyslipidemia effects other organs simultaneously is unknown. We set out to discover how the liver responds to a dyslipidemic insult through the creation of inducible mouse models based on human familial hypercholesterolemia mutations and in vivo tracing of APOB. An acute transition to atherogenic APOB-lipoprotein plasma levels resulted in rapid accumulation of triglycerides and cholesterol in the liver. Single cell RNA-seq and flow cytometry disclosed that multiple immune cells have the ability to engulf APOB-lipoproteins. However bulk RNA-seq of the liver revealed an inflammatory Kupffer cell-specific transcriptional program that could not be activated by a western diet alone. Depletion of Kupffer cells through clodronate liposomes or CD8 T cell targeting rapidly raised plasma lipoprotein levels, indicating that these liver macrophages help restrain and buffer atherogenic lipoproteins, whilst simultaneously secreting pro-atherosclerotic factors into plasma. Our results place Kupffer cells as a key gateway in organizing systemic responses at the initiation of atherosclerosis.

immunology↗

bioRxiv2021

Straintables: An application that extracts sequences fromgenome assemblies and generates dissimilarity matrices

Background and ObjectivesThe dissimilarity matrix (DM) is an important component of phylogenetic analysis, and many software packages exist to build and show DMs. However, as the common input for this type of software are sequences in FASTA file format, the process of extracting and aligning each set of sequences to produce a big number of matrices can be laborious. Additionally, existing software do not facilitate the comparison of clusters of similarity across several DMs built for the same group of individuals, using different genomic regions. To address our requirements of such a tool, we designed Straintables to extract specific genomic region sequences from a group of intraspecies genomic assemblies, using extracted sequences to build dissimilarity matrices. MethodsA Python module with executable scripts was developed for a study on genetic diversity across strains of Toxoplasma gondii, being a general purpose system for DM calculation and visualization for preliminary phylogenetic studies. For automatic region sequence extraction from genomic assemblies we assembled a system that designs virtual primers using reference sequences located at genomic annotations, then matches those primers on genome files by using regex patterns. Extracted sequences are then aligned using Clustal Omega and compared to generate matrices. ResultsUsing this software saves the user from manual preparation and alignment of the sequences, a process that can be laborious when a large number of assemblies or regions are involved. The automatic sequence extraction process can be checked against BLAST results using extracted sequence as queries, where correct results were observed for same-species pools for various organisms. The package also contains a matrix visualization tool focused on cluster visualization, capable of drawing matrices into image files with custom settings, and features methods of reordering matrices to facilitate the comparison of clustering patterns across two or more matrices. ConclusionStraintables may replace and extend the functionality of existing matrix-oriented phylogenetic software, featuring automatic region extraction from genomic assemblies and enhanced matrix visualization capabilities emphasizing cluster identification. This module is open source, available at GitHub (https://github.com/Gab0/straintables) under a MIT license and also as a PIPY package. HighlightsSimple in-silico protocol for generation, visualization and comparison of dissimilarity matrices. Accurate automatic sequence extraction from multiple genomic assemblies by using virtual primers built from reference sequences in an annotation file. Draws matrices as images, with enhanced cluster visualization and customized options. Supports reordering of matrix indices to better visualize clustering pattern conservation across multiple regions.

bioinformatics↗

bioRxiv2020

Resource reallocation in engineered Escherichia coli strains with reduced genomes

A major challenge in synthetic biology is properly balancing evolved and engineered functions without compromising microbial fitness. Many microbial proteins are not required for growth in regular laboratory conditions, but it is unclear what fraction of the proteome can be eliminated to increase bioproduction and maintain fitness. Here, we investigated the effects of massive genome reduction in E. coli on the expression level and evolutionary stability of a model biosynthetic pathway to produce the pigment protodeoxyviolacein (PDV). We identified an amino acid metabolism imbalance and compromised growth that were correlated with elimination of genes associated with significant proteome fraction. Proteomic profiling suggested that increased amino acid pools are responsible for an alleviation of fitness defects associated with PDV expression. In addition, all strains with genome reductions that significantly affected the proteome exhibited decreased stability of PDV production compared to the wild-type strain under persistent PDV expression conditions despite the alleviation of fitness defects. These findings exhibit the importance of balancing evolved functions with engineered ones to achieve an optimal balance of fitness and bioproduction.

bioengineering↗

bioRxiv2020

The SAGE genetic toolkit enables highly efficient, iterative site-specific genome engineering in bacteria

Sustainable enhancements to crop productivity and increased resilience to adverse conditions are critical for modern agriculture, and application of plant growth promoting rhizobacteria (PGPR) is a promising method to achieve these goals. However, many desirable PGPR traits are highly regulated in their native microbe, limited to certain plant rhizospheres, or insufficiently active for agricultural purposes. Synthetic biology can address these limitations, but its application is limited by availability of appropriate tools for sophisticated, high-throughput genome engineering that function in environments where selection for DNA maintenance is impractical. Here we present an orthogonal, Serine-integrase Assisted Genome Engineering (SAGE) system, which enables iterative, site-specific integration of up to 10 different DNA constructs at efficiency on par or better than replicating plasmids. SAGE does not require use of replicating plasmids to deliver recombination machinery, and employs a secondary serine-integrase to excise and recycle selection markers. Furthermore, unlike the widely utilized pBBR1 origin, DNA transformed using SAGE is stable without selection. We highlight SAGEs utility by constructing a 287-member constitutive promoter library with a [~]40,000-fold dynamic range in P. fluorescens SBW25. We show that SAGE functions robustly in diverse - and {gamma}-proteobacteria, thus providing evidence that it will be broadly useful for engineering industrial or environmental bacteria.

synthetic biology↗

bioRxiv2020

High speed large scale automated isolation of SARS-CoV-2 from clinical samples using miniaturized co-culture coupled with high content screening

SARS-CoV-2, a novel coronavirus infecting humans, is responsible for the current COVID-19 global pandemic. If several strains could be isolated worldwide, especially for in-vitro drug susceptibility testing and vaccine development, few laboratories routinely isolate SARS-CoV-2. This is due to the fact that the current co-culture strategy is highly time consuming and requires working in a biosafety level 3 laboratory. In this work, we present a new strategy based on high content screening automated microscopy (HCS) allowing large scale isolation of SARS-CoV-2 from clinical samples in 1 week. A randomized panel of 104 samples, including 72 tested positive by RT-PCR and 32 tested negative, were processed with our HCS procedure and were compared to the classical isolation procedure. Isolation rate was 43 % with both strategies on RT-PCR positive samples, and was correlated with the initial RNA viral load in the samples, where we obtained a positivity threshold of 27 Ct. Co-culture delays were shorter with HCS strategy, where 80 % of the positive samples were recovered by the third day of co-culture, as compared to only 25 % with the classic strategy. Moreover, only the HCS strategy allowed us to recover all the positive elements after 1 week of co-culture. This system allows rapid and automated screening of clinical samples with minimal operator work load, thus reducing the risks of contamination.

microbiology↗

bioRxiv2020

Advantages and limits of metagenomic assembly and binning of a giant virus

Giant viruses have large genomes, often within the size range of cellular organisms. This distinguishes them from most other viruses and demands additional effort for the successful recovery of their genomes from environmental sequence data. Here we tested the performance of genome-resolved metagenomics on a recently isolated giant virus, Fadolivirus, by spiking it into an environmental sample from which two other giant viruses were isolated. At high spike-in levels, metagenome assembly and binning led to the successful genomic recovery of Fadolivirus from the sample. A complementary survey of viral hallmark genes indicated the presence of other giant viruses in the sample matrix, but did not detect the two isolated from this sample. Our results indicate that genome-resolved metagenomics is a valid approach for the recovery of near-complete giant virus genomes given that sufficient clonal particles are present. Our data also underline that a vast majority of giant viruses remain currently undetected, even in an era of terabase-scale metagenomics.

microbiology↗

bioRxiv2019

High Content Screening, a reliable system for Coxiella burnetii isolation from clinical samples

Q fever, caused by Coxiella burnetii, is a worldwide zoonotic disease that may cause severe forms in humans and requires a specific and prolonged antibiotic treatment. Although the current serological and molecular detection tools enable a reliable diagnosis of the disease, culture of C. burnetii strains is mandatory to evaluate their antibiotic susceptibility and sequence their genome in order to optimize patient management and epidemiological studies. However, cultivating this fastidious microorganism is difficult and restricted to reference centers as it requires biosafety-level 3 laboratories and relies on cell culture performed by experienced technicians. In addition, the culture yield is low, which results in a small number of isolates being available. In this work, we developed a novel high content screening (HCS) isolation strategy based on optimized high-throughput cell culture and automated microscopic detection of infected cells with specifically-designed algorithms targeting cytopathic effects. This method was more efficient than the shell-vial assay when applied to both frozen specimens (7 isolates recovered by HCS only, sensitivity 91% vs 78% for shell-vial) and fresh samples (1 additional isolate using HCS, sensitivity 7% vs 5% for shell-vial). In addition, detecting positive cultures by an automated microscope reduced the need for expertise and saved 24% of technician working time. Application of HCS to antibiotic susceptibility testing of 12 strains demonstrated that it was as efficient as the standard procedure that combines shell-vial culture and quantitative PCR. Overall, this high-throughput HCS system paves the way to the development of improved cell culture isolation of human viruses.

microbiology↗