Engineering selectivity of Cutibacterium acnes phages by epigenetic imprinting.

Cutibacterium acnes (C. acnes) is a gram-positive bacterium and a member of the human skin microbiome. Despite being the most abundant skin commensal, certain members have been associated with common inflammatory disorders such as acne vulgaris. The availability of the complete genome sequences from various C. acnes clades have enabled the identification of putative methyltransferases, some of them potentially belonging to restriction-modification (R-M) systems which protect the host of invading DNA. However, little is known on whether these systems are functional in the different C. acnes strains. To investigate the activity of these putative R-M and their relevance in host protective mechanisms, we analyzed the methylome of six representative C. acnes strains by Oxford Nanopore Technologies (ONT) sequencing. We detected the presence of a 6-methyladenine modification at a defined DNA consensus sequence in strain KPA171202 and recombinant expression of this R-M system confirmed its methylation activity. Additionally, a R-M knockout mutant verified the loss of methylation properties of the strain. We studied the potential of one C. acnes bacteriophage (PAD20) in killing various C. acnes strains and linked an increase in its specificity to phage DNA methylation acquired upon infection of a methylation competent strain. We demonstrate a therapeutic application of this mechanism where phages propagated in R-M deficient strains selectively kill R-M deficient acne-prone clades while probiotic ones remain resistant to phage infection.

CRISPR-Analytics (CRISPR-A): A platform for precise analytics and simulations for gene editing.

Gene editing characterization with currently available tools does not always give precise relative proportions among the different types of gene edits present in an edited bulk of cells. We have developed CRISPR-Analytics, CRISPR-A, which is a comprehensive and versatile genome editing web application tool and a nextflow pipeline to give support to gene editing experimental design and analysis. CRISPR-A provides a robust gene editing analysis pipeline composed of data analysis tools and simulation. It achieves higher accuracy than current tools and expands the functionality. The analysis includes mock-based noise correction, spike-in calibrated amplification bias reduction, and advanced interactive graphics. This expanded robustness makes this tool ideal for analyzing highly sensitive cases such as clinical samples or experiments with low editing efficiencies. It also provides an assessment of experimental design through the simulation of gene editing results. Therefore, CRISPR-A is ideal to support multiple kinds of experiments such as double-stranded DNA break-based engineering, base editing (BE), primer editing (PE), and homology-directed repair (HDR), without the need of specifying the used experimental approach.

Enabling large-scale genome editing at repetitive elements by reducing DNA nicking.

To extend the frontier of genome editing and enable editing of repetitive elements of mammalian genomes, we made use of a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks and single-strand breaks. We used a set of gRNAs targeting repetitive elements-ranging in target copy number from about 32 to 161 000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ∼13 200 and ∼12 200 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.

Safety and Efficacy of Topically Applied Selected Cutibacterium acnes Strains over Five Weeks in Patients with Acne Vulgaris: An Open-label, Pilot Study.

Imbalance in skin microflora, particularly related to certain Cutibacterium acnes strains, may trigger acne. Application of non-acne-causing strains to the skin may modulate the skin microbiome and thereby lead to a reduction in acne. This pilot study evaluates the safety and efficacy of microbiome modulation on acne-prone skin. The study had 2 phases: active induction (5% benzoyl peroxide gel, 7 days) and interventional C. acnes strains treatment (5 weeks). Patients were randomized to either topical skin formulations PT1 (2 strains of C. acnes Single Locus Sequence Typing [SLST] type C3 and K8, 50% each) or PT2 (4 strains of C. acnes SLST type C3 [55%], K8 [5%], A5 [30%] and F4 [10%]). Safety and efficacy was evaluated in 14 patients (PT1=8/14, PT2=6/14). Skin microbiome composition shifted towards study formulations. No untoward adverse events, visible irritation, or significant flare-up were observed. Non-inflamed lesions and skin pH were reduced. Comedone counts improved clinically with no deterioration in inflammatory lesions.

Genome editing assessment using CRISPR Genome Analyzer (CRISPR-GA).

SUMMARY: Clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies have revolutionized human genome engineering and opened countless possibilities to basic science, synthetic biology and gene therapy. Albeit the enormous potential of these tools, their performance is far from perfect. It is essential to perform a posterior careful analysis of the gene editing experiment. However, there are no computational tools for genome editing assessment yet, and current experimental tools lack sensitivity and flexibility. We present a platform to assess the quality of a genome editing experiment only with three mouse clicks. The method evaluates next-generation data to quantify and characterize insertions, deletions and homologous recombination. CRISPR Genome Analyzer provides a report for the locus selected, which includes a quantification of the edited site and the analysis of the different alterations detected. The platform maps the reads, estimates and locates insertions and deletions, computes the allele replacement efficiency and provides a report integrating all the information. AVAILABILITY AND IMPLEMENTATION: CRISPR-GA Web is available at http://crispr-ga.net. Documentation on CRISPR-GA instructions can be found at http://crispr-ga.net/documentation.html CONTACT: mguell@genetics.med.harvard.edu.

Optimization of scarless human stem cell genome editing.

Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7-8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, we demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks.

Delivery of a sebum modulator by an engineered skin microbe in mice.

Microorganisms can be equipped with synthetic genetic programs for the production of targeted therapeutic molecules. Cutibacterium acnes is the most abundant commensal of the human skin, making it an attractive chassis to create skin-delivered therapeutics. Here, we report the engineering of this bacterium to produce and secrete the therapeutic molecule neutrophil gelatinase-associated lipocalin, in vivo, for the modulation of cutaneous sebum production.

Strand-specific deep sequencing of the transcriptome.

Several studies support that antisense-mediated regulation may affect a large proportion of genes. Using the Illumina next-generation sequencing platform, we developed DSSS (direct strand specific sequencing), a strand-specific protocol for transcriptome sequencing. We tested DSSS with RNA from two samples, prokaryotic (Mycoplasma pneumoniae) as well as eukaryotic (Mus musculus), and obtained data containing strand-specific information, using single-read and paired-end sequencing. We validated our results by comparison with a strand-specific tiling array data set for strain M129 of the simple prokaryote M. pneumoniae, and by quantitative PCR (qPCR). The results of DSSS were very well supported by the results from tiling arrays and qPCR. Moreover, DSSS provided higher dynamic range and single-base resolution, thus enabling efficient antisense detection and the precise mapping of transcription start sites and untranslated regions. DSSS data for mouse confirmed strand specificity of the protocol and the general applicability of the approach to studying eukaryotic transcription. We propose DSSS as a simple and efficient strategy for strand-specific transcriptome sequencing and as a tool for genome annotation exploiting the increased read lengths that next-generation sequencing technology now is capable to deliver.

Transcription start site associated RNAs in bacteria.

Here, we report the genome-wide identification of small RNAs associated with transcription start sites (TSSs), termed tssRNAs, in Mycoplasma pneumoniae. tssRNAs were also found to be present in a different bacterial phyla, Escherichia coli. Similar to the recently identified promoter-associated tiny RNAs (tiRNAs) in eukaryotes, tssRNAs are associated with active promoters. Evidence suggests that these tssRNAs are distinct from previously described abortive transcription RNAs. ssRNAs have an average size of 45 bases and map exactly to the beginning of cognate full-length transcripts and to cryptic TSSs. Expression of bacterial tssRNAs requires factors other than the standard RNA polymerase holoenzyme. We have found that the RNA polymerase is halted at tssRNA positions in vivo, which may indicate that a pausing mechanism exists to prevent transcription in the absence of genes. These results suggest that small RNAs associated with TSSs could be a universal feature of bacterial transcription.

Establishing a Cell-Free Transcription-Translation Platform for Cutibacterium acnes to Prototype Engineered Metabolic and Synthetic Biology.

In the past few years, new bacterial-cell-free transcription-translation systems have emerged as potent and quick platforms for protein production as well as for prototyping of DNA regulatory elements, genetic circuits, and metabolic pathways. The Gram-positive commensal Cutibacterium acnes is one of the most abundant bacteria present in the human skin microbiome. However, it has recently been reported that some C. acnes phylotypes can be associated with common inflammatory skin conditions, such as acne vulgaris, whereas others seem to play a protective role, acting as possible "skin probiotics". This fact has made C. acnes become a bacterial model of interest for the cosmetic industry. In the present study we report for the first time the development and optimization of a C. acnes-based cell-free system (CFS) that is able to produce 85 µg/mL firefly luciferase. We highlight the importance of harvesting the bacterial pellet in mid log phase and maintaining CFS reactions at 30 °C and physiological pH to obtain the optimal yield. Additionally, a C. acnes promoter library was engineered to compare coupled in vitro TX-TL activities, and a temperature biosensor was tested, demonstrating the wide range of applications of this toolkit in the synthetic biology field.

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

Cutibacterium acnes (C. acnes) is one of the most prevalent bacteria that forms the human skin microbiota. Specific phylotypes of C. acnes have been associated with the development of acne vulgaris, while other phylotypes have been linked to healthy skin. In this scenario, bacterial extracellular vesicles (EVs) play a role in the interkingdom communication role with the human host. The purpose of this study was to examine the impact of EVs generated by various phylotypes of C. acnes on inflammation and sebum production using different in vitro skin cell types. The main findings of this study reveal that the proteomic profile of the cargo embodied in the EVs reflects distinct characteristics of the different C. acnes phylotypes in terms of life cycle, survival, and virulence. The in vitro skin cell types showed an extended pro-inflammatory modulation of SLST A1 EVs consistently triggering the activation of the inflammation-related factors IL-8, IL-6, TNFα and GM-CSF, in comparison to SLST H1 and SLST H2. Additionally, an acne-prone skin model utilizing PCi-SEB and arachidonic acid as a sebum inducer, was employed to investigate the impact of C. acnes EVs on sebum regulation. Our findings indicated that all three types of EVs significantly inhibited sebum production after a 24-h treatment period, with SLST H1 EVs exhibiting the most pronounced inhibitory effect when compared to the positive control. The results of this study highlight the protective nature of C. acnes SLST H1 EVs and their potential use as a natural treatment option for alleviating symptoms associated with inflammation and oily skin.

Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR-Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.

Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi.

The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.

Quantification of mRNA and protein and integration with protein turnover in a bacterium.

Biological function and cellular responses to environmental perturbations are regulated by a complex interplay of DNA, RNA, proteins and metabolites inside cells. To understand these central processes in living systems at the molecular level, we integrated experimentally determined abundance data for mRNA, proteins, as well as individual protein half-lives from the genome-reduced bacterium Mycoplasma pneumoniae. We provide a fine-grained, quantitative analysis of basic intracellular processes under various external conditions. Proteome composition changes in response to cellular perturbations reveal specific stress response strategies. The regulation of gene expression is largely decoupled from protein dynamics and translation efficiency has a higher regulatory impact on protein abundance than protein turnover. Stochastic simulations using in vivo data show how low translation efficiency and long protein half-lives effectively reduce biological noise in gene expression. Protein abundances are regulated in functional units, such as complexes or pathways, and reflect cellular lifestyles. Our study provides a detailed integrative analysis of average cellular protein abundances and the dynamic interplay of mRNA and proteins, the central biomolecules of a cell.

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

Comprehensive characterisation of genome engineering technologies is relevant for their development and safe use in human gene therapy. Short-read based methods can overlook insertion events in repetitive regions. We develop INSERT-seq, 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. The experimental pipeline improves the number of mappable insertions at repetitive regions by 4.8-7.3% and larger repeats are processed with a computational peak calling pipeline. INSERT-seq is a simple, cheap and robust method to quantitatively characterise DNA integration in diverse ex vivo and in vivo samples.

DNA Damage Protection for Enhanced Bacterial Survival Under Simulated Low Earth Orbit Environmental Conditions in Escherichia coli.

Some organisms have shown the ability to naturally survive in extreme environments, even outer space. Some of these have natural mechanisms to resist severe DNA damage from conditions such as ionizing and non-ionizing radiation, extreme temperatures, and low pressures or vacuum. A good example can be found in Deinococcus radiodurans, which was exposed to severe conditions such as those listed in the Exposure Facility of the International Space Station (ISS) for up to three years. Another example are tardigrades (Ramazzottius varieornatus) which are some of the most resilient animals known. In this study, the survival under simulated Low earth Orbit (LEO) environmental conditions was tested in Escherichia coli. The radiation resistance of this bacteria was enhanced using the Dsup gene from R. varieornatus, and two more genes from D. radiodurans involved in DNA damage repair, RecA and uvrD. The enhanced survival to wide ranges of temperatures and low pressures was then tested in the new strains. This research constitutes a first step in the creation of new bacterial strains engineered to survive severe conditions and adapting existing species for their survival in remote environments, including extra-terrestrial habitats. These strains could be key for the development of environments hospitable to life and could be of use for ecological restoration and space exploration. In addition, studying the efficacy and the functioning of the DNA repair mechanisms used in this study could be beneficial for medical and life sciences engineering.

Skin microbiome transplantation and manipulation: Current state of the art.

Many skin conditions are associated with an imbalance in the skin microbiome. In recent years, the skin microbiome has become a hot topic, for both therapeutic and cosmetic purposes. The possibility of manipulating the human skin microbiome to address skin conditions has opened exciting new paths for therapy. Here we review the skin microbiome manipulation strategies, ranging from skin microbiome transplantation, over skin bacteriotherapy to the use of prebiotics, probiotics and postbiotics. We summarize all efforts undertaken to exchange, manipulate, transplant or selectively apply the skin microbiome to date. Multiple microbial groups have been targeted, since they have been proven to be beneficial for skin health. We focus on the most common skin disorders and their associated skin microbiome dysbiosis and we review the existing scientific data and clinical trials undertaken to combat these skin conditions. The skin microbiome represents a novel platform for therapy. Transplantation of a complete microbiome or application of single strains has demonstrated beneficial therapeutic application.

From Dysbiosis to Healthy Skin: Major Contributions of Cutibacterium acnes to Skin Homeostasis.

Cutibacterium acnes is the most abundant bacterium living in human, healthy and sebum-rich skin sites, such as the face and the back. This bacterium is adapted to this specific environment and therefore could have a major role in local skin homeostasis. To assess the role of this bacterium in healthy skin, this review focused on (i) the abundance of C. acnes in the skin microbiome of healthy skin and skin disorders, (ii) its major contributions to human skin health, and (iii) skin commensals used as probiotics to alleviate skin disorders. The loss of C. acnes relative abundance and/or clonal diversity is frequently associated with skin disorders such as acne, atopic dermatitis, rosacea, and psoriasis. C. acnes, and the diversity of its clonal population, contributes actively to the normal biophysiological skin functions through, for example, lipid modulation, niche competition and oxidative stress mitigation. Compared to gut probiotics, limited dermatological studies have investigated skin probiotics with skin commensal strains, highlighting their unexplored potential.