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PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics.Nature Communications Jun 2024The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a...
The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.
Topics: Humans; Gene Editing; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Francisella; Bacterial Proteins; Leber Congenital Amaurosis; Streptococcus pyogenes; HEK293 Cells; Mutation; RNA, Guide, CRISPR-Cas Systems; Protein Engineering; Genome, Human
PubMed: 38942756
DOI: 10.1038/s41467-024-49233-w -
Journal of Oral Biosciences Jun 2024This study aimed to investigate the regulatory mechanisms governing dental mesenchymal cell commitment during tooth development, focusing on odontoblast differentiation...
Exploring the Role of DNMT1 in Dental Papilla Cell Fate Specification during Mouse Tooth Germ Development through Integrated Single-Cell Transcriptomics and Bulk RNA Sequencing.
OBJECTIVES
This study aimed to investigate the regulatory mechanisms governing dental mesenchymal cell commitment during tooth development, focusing on odontoblast differentiation and the role of epigenetic regulation in this process.
METHODS
We performed single-cell RNA sequencing (scRNA-seq) of dental cells from embryonic day 14.5 (E14.5) mice to understand the heterogeneity of developing tooth germ cells. Computational analyses including gene regulatory network (GRN) assessment were conducted. We validated our findings using immunohistochemistry (IHC) and in vitro loss-of-function analyses using the DNA methyltransferase 1 (DNMT1) inhibitor Gsk-3484862 in primary dental mesenchymal cells (DMCs) isolated from E14.5 mouse tooth germs. Bulk RNA-seq of Gsk-3484862-treated DMCs was performed to identify potential downstream targets of DNMT1.
RESULTS
scRNA-seq analysis revealed diverse cell populations within the tooth germs, including epithelial, mesenchymal, immune, and muscle cells. Using single-cell regulatory network inference and clustering (SCENIC), we identified Dnmt1 as a key regulator of early odontoblast development. IHC analysis showed the ubiquitous expression of DNMT1 in the dental papilla and epithelium. Bulk RNA-seq of cultured DMCs showed that Gsk-3484862 treatment upregulated odontoblast-related genes, whereas genes associated with cell division and the cell cycle were downregulated. Integrated analysis of bulk RNA-seq data with scRNA-seq SCENIC profiles was used to identify the potential Dnmt1 target genes.
CONCLUSIONS
Dnmt1 may negatively affect odontoblast commitment and differentiation during tooth development. These findings contribute to a better understanding of the molecular mechanisms underlying tooth development and future development of hard-tissue regenerative therapies.
PubMed: 38942194
DOI: 10.1016/j.job.2024.06.010 -
Cryobiology Jun 2024Growing concerns over declining male semen quality and rising infertility have shifted attention to male fertility. Sperm cryopreservation emerges as a crucial tool in...
Growing concerns over declining male semen quality and rising infertility have shifted attention to male fertility. Sperm cryopreservation emerges as a crucial tool in preserving male fertility, especially for patients who need proactive preservation, such as cancer patients before undergoing radiation or chemotherapy. Although cryopreservation does not directly address infertility, effective preservation can support future fertility. However, the process may compromise sperm DNA integrity. Despite their impairment, damaged sperm often retain vitality and may still have the potential to fertilize an egg. Nonetheless, if damaged sperm fertilize an egg, excessive DNA damage could impede embryo implantation and development, despite the egg's repair capabilities. Consequently, precise detection of sperm DNA damage is crucial and urgent. To better address the issue of sperm DNA damage detection, we have introduced a novel fluorescence biosensor technology known as the TDT/SD Probe. This technology utilizes terminal deoxynucleotidyl transferase (TdT) and strand displacement probes to accurately detect the number of sperm DNA breakage points during the cryopreservation process. Experimental results reveal that the number of sperm DNA breakpoints significantly increases after both sperm vitrification (8.17×10) and conventional slow freezing (10.80×10), compared to the DNA breakpoints of fresh semen samples (5.19×10). However, sperm vitrification has the least impact on sperm breakage points. This research provides innovative means for further optimizing sperm preservation techniques by offering a novel DNA damage detection method, enabling more precise assessment of sperm DNA damage during the freezing process.
PubMed: 38942068
DOI: 10.1016/j.cryobiol.2024.104937 -
MBio Jun 2024Conjugative type 4 secretion systems (T4SSs) are the main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. To deliver the DNA...
Conjugative type 4 secretion systems (T4SSs) are the main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. To deliver the DNA substrate to recipient cells, it must cross the cell envelopes of both donor and recipient bacteria. In the T4SS from the enterococcal conjugative plasmid pCF10, PrgK is known to be the active cell wall degrading enzyme. It has three predicted extracellular hydrolase domains: metallo-peptidase (LytM), soluble lytic transglycosylase (SLT), and cysteine, histidine-dependent amidohydrolases/peptidases (CHAP). Here, we report the structure of the LytM domain and show that its active site is degenerate and lacks the active site metal. Furthermore, we show that only the predicted SLT domain is functional and that it unexpectedly has a muramidase instead of a lytic transglycosylase activity. While we did not observe any peptidoglycan hydrolytic activity for the LytM or CHAP domain, we found that these domains downregulated the SLT muramidase activity. The CHAP domain was also found to be involved in PrgK dimer formation. Furthermore, we show that PrgK interacts with PrgL, which likely targets PrgK to the rest of the T4SS. The presented data provides important information for understanding the function of Gram-positive T4SSs.IMPORTANCEAntibiotic resistance is a large threat to human health and is getting more prevalent. One of the major contributors to the spread of antibiotic resistance among different bacteria is type 4 secretion systems (T4SS). However, mainly T4SSs from Gram-negative bacteria have been studied in detail. T4SSs from Gram-positive bacteria, which stand for more than half of all hospital-acquired infections, are much less understood. The significance of our research is in identifying the function and regulation of a cell wall hydrolase, a key component of the pCF10 T4SS from . This system is one of the best-studied Gram-positive T4SSs, and this added knowledge aids in our understanding of horizontal gene transfer in as well as other medically relevant Gram-positive bacteria.
PubMed: 38940556
DOI: 10.1128/mbio.00488-24 -
Bioanalysis Jun 2024Increased knowledge of biodistribution and pharmacokinetics of lipid nanoparticle (LNP)-encapsulated mRNA drug components may aid efficacy and safety evaluation. Mice...
Increased knowledge of biodistribution and pharmacokinetics of lipid nanoparticle (LNP)-encapsulated mRNA drug components may aid efficacy and safety evaluation. Mice were subcutaneously administrated LNP encapsulated enhanced green fluorescent protein mRNA and sampled up to 72 h after dosing. LNP, mRNA and translated protein were quantified by LC-MS, branched DNA and ELISA. Highest levels of LNP and mRNA were detected in skin, followed by spleen, but also rapidly distributed to circulation. Translated protein showed high concentration in skin and spleen, but also in liver and kidney across 24 h where the LNP was cleared at 4 h. Subcutaneously dosing LNP encapsulated mRNA in mice resulted in a nonlinear relationship of LNP, mRNA and protein concentration across multiple tissues.
PubMed: 38940441
DOI: 10.1080/17576180.2024.2360361 -
Frontiers in Bioscience (Landmark... May 2024Epigenetics refers to heritable changes in gene expression and function that impact nuclear processes associated with chromatin, all without altering DNA sequences.... (Review)
Review
Epigenetics refers to heritable changes in gene expression and function that impact nuclear processes associated with chromatin, all without altering DNA sequences. These epigenetic patterns, being heritable traits, are vital biological mechanisms that intricately regulate gene expression and heredity. The application of chemical labeling and single-cell resolution mapping strategies has significantly facilitated large-scale epigenetic modifications in nucleic acids over recent years. Notably, epigenetic modifications can induce heritable phenotypic changes, regulate cell differentiation, influence cell-specific gene expression, parentally imprint genes, activate the X chromosome, and stabilize genome structure. Given their reversibility and susceptibility to environmental factors, epigenetic modifications have gained prominence in disease diagnosis, significantly impacting clinical medicine research. Recent studies have uncovered strong links between epigenetic modifications and the pathogenesis of metabolic cardiovascular diseases, including congenital heart disease, heart failure, cardiomyopathy, hypertension, and atherosclerosis. In this review, we provide an overview of the progress in epigenetic research within the context of cardiovascular diseases, encompassing their pathogenesis, prevention, diagnosis, and treatment. Furthermore, we shed light on the potential prospects of nucleic acid epigenetic modifications as a promising avenue in clinical medicine and biomedical applications.
Topics: Humans; Epigenesis, Genetic; Cardiovascular Diseases; DNA Methylation; Animals
PubMed: 38940023
DOI: 10.31083/j.fbl2906205 -
Chemical Science Jun 2024Study of alternating DNA GC sequences by different time-resolved spectroscopies has provided fundamental information on the interaction between UV light and DNA, a...
Study of alternating DNA GC sequences by different time-resolved spectroscopies has provided fundamental information on the interaction between UV light and DNA, a process of great biological importance. Multiple decay paths have been identified, but their interplay is still poorly understood. Here, we characterize the photophysics of GC-DNA by integrating different computational approaches, to study molecular models including up to 6 bases described at a full quantum mechanical level. Quantum dynamical simulations, exploiting a nonadiabatic linear vibronic coupling (LVC) model, coupled with molecular dynamics sampling of the initial structures of a (GC) DNA duplex, provide new insights into the photophysics in the sub-picosecond time-regime. They indicate a substantial population transfer, within 50 fs, from the spectroscopic states towards G → C charge transfer states involving two stacked bases (CT), thus explaining the ultrafast disappearance of fluorescence. This picture is consistent with that provided by quantum mechanical geometry optimizations, using time dependent-density functional theory and a polarizable continuum model, which we use to parametrize the LVC model and to map the main excited state deactivation pathways. For the first time, the infrared and excited state absorption signatures of the various states along these pathways are comprehensively mapped. The computational models suggest that the main deactivation pathways, which, according to experiment, lead to ground state recovery on the 10-50 ps time scale, involve CT followed by interstrand proton transfer from the neutral G to C. Our calculations indicate that CT is populated to a larger extent and more rapidly in GC than in CG steps and suggest the likely involvement of monomer-like and interstrand charge transfer decay routes for isolated and less stacked CG steps. These findings underscore the importance of the DNA sequence and thermal fluctuations for the dynamics. They will also aid the interpretation of experimental results on other sequences.
PubMed: 38939156
DOI: 10.1039/d4sc00910j -
Molecular Therapy : the Journal of the... Jun 2024Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. Conventional gene editing platforms that rely on nuclease...
Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. Conventional gene editing platforms that rely on nuclease activity, such as Clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point™ base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared to the conventional CRISPR-Cas9 system. Site-specific knock-in of a chimeric antigen receptor (CAR) and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.
PubMed: 38937969
DOI: 10.1016/j.ymthe.2024.06.033 -
BMC Plant Biology Jun 2024Caryodaphnopsis, a group of tropical trees (ca. 20 spp.) in the family Lauraceae, has an amphi-Pacific disjunct distribution: ten species are distributed in Southeast...
BACKGROUND
Caryodaphnopsis, a group of tropical trees (ca. 20 spp.) in the family Lauraceae, has an amphi-Pacific disjunct distribution: ten species are distributed in Southeast Asia, while eight species are restricted to tropical rainforests in South America. Previously, phylogenetic analyses using two nuclear markers resolved the relationships among the five species from Latin America. However, the phylogenetic relationships between the species in Asia remain poorly known.
RESULTS
Here, we first determined the complete mitochondrial genome (mitogenome), plastome, and the nuclear ribosomal cistron (nrDNA) sequences of C. henryi with lengths of 1,168,029 bp, 154,938 bp, and 6495 bp, respectively. We found 2233 repeats and 368 potential SSRs in the mitogenome of C. henryi and 50 homologous DNA fragments between its mitogenome and plastome. Gene synteny analysis revealed a mass of rearrangements in the mitogenomes of Magnolia biondii, Hernandia nymphaeifolia, and C. henryi and only six conserved clustered genes among them. In order to reconstruct relationships for the ten Caryodaphnopsis species in Asia, we created three datasets: one for the mitogenome (coding genes and ten intergenic regions), another for the plastome (whole genome), and the other for the nuclear ribosomal cistron. All of the 22 Caryodaphnopsis individuals were divided into four, five, and six different clades in the phylogenies based on mitogenome, plastome, and nrDNA datasets, respectively.
CONCLUSIONS
The study showed phylogenetic conflicts within and between nuclear and organellar genome data of Caryodaphnopsis species. The sympatric Caryodaphnopsis species in Hekou and Malipo SW China may be related to the incomplete lineage sorting, chloroplast capture, and/or hybridization, which mixed the species as a complex in their evolutionary history.
Topics: Phylogeny; Genome, Mitochondrial; Lauraceae; Genome, Plant
PubMed: 38937691
DOI: 10.1186/s12870-024-05050-3 -
Neurocritical Care Jun 2024Hypoxic-ischemic brain injury is a common cause of mortality after cardiac arrest (CA) and cardiopulmonary resuscitation; however, the specific underlying mechanisms are...
BACKGROUND
Hypoxic-ischemic brain injury is a common cause of mortality after cardiac arrest (CA) and cardiopulmonary resuscitation; however, the specific underlying mechanisms are unclear. This study aimed to explore postresuscitation changes based on multi-omics profiling.
METHODS
A CA swine model was established, and the neurological function was assessed at 24 h after resuscitation, followed by euthanizing animals. Their fecal, blood, and hippocampus samples were collected to analyze gut microbiota, metabolomics, and transcriptomics.
RESULTS
The 16S ribosomal DNA sequencing showed that the microbiota composition and diversity changed after resuscitation, in which the abundance of Akkermansia and Muribaculaceae_unclassified increased while the abundance of Bifidobacterium and Romboutsia decreased. A relationship was observed between CA-related microbes and metabolites via integrated analysis of gut microbiota and metabolomics, in which Escherichia-Shigella was positively correlated with glycine. Combined metabolomics and transcriptomics analysis showed that glycine was positively correlated with genes involved in apoptosis, interleukin-17, mitogen-activated protein kinases, nuclear factor kappa B, and Toll-like receptor signal pathways.
CONCLUSIONS
Our results provided novel insight into the mechanism of hypoxic-ischemic brain injury after resuscitation, which is envisaged to help identify potential diagnostic and therapeutic markers.
PubMed: 38937417
DOI: 10.1007/s12028-024-02038-7