Search

Hypoxia enhances histone methylation by inhibiting oxygen- and α-ketoglutarate-dependent demethylases, resulting in increased methylated histones. This study reveals how hypoxia-induced methylation affects histone clipping and the reorganization of heterochromatin into senescence-associated heterochromatin foci (SAHF) during oncogene-induced senescence (OIS) in IMR90 human fibroblasts. Notably, using top-down proteomics, we discovered specific cleavage sites targeted by Cathepsin L (CTSL) in H3, H2B and H4 during Raf activation, identifying novel sites in H2B and H4. Hypoxia counteracts CTSL-mediated histone clipping by promoting methylation without affecting CTSL's activity. This increase in methylation under hypoxia protects against clipping, reshaping the epigenetic landscape and influencing chromatin accessibility, as shown by ATAC-seq analysis. These insights underscore the pivotal role of hypoxia-induced histone methylation in protecting chromatin from significant epigenetic shifts during cellular aging., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We investigated the regulation of histone deacetylases (HDACs) by miR-2861 in the osteoblastic differentiation of human mesenchymal stem cells (MSCs) and miR-2861 binding site by CRISPR activation (CRISPRa). Transfection of miR-2861 into human MSCs was performed and the effect on osteoblast differentiation was analyzed. Using catalytically inactive Cas12a, the CRISPRa system induced targeted overexpression of endogenous miRNA and repressed the luciferase activities of reporters that contained functional miRNA target sites. The delivery of miR-2861 into MSCs enhanced osteoblast differentiation by decreased expressions of the HDAC1, 4 and 5 genes. The mechanism of HDAC5 repression by miR-2861 in humans has not been fully elucidated. To this end, the HDAC5 mRNA sequence was analyzed and a putative primate-specific miR-2861 binding site was identified in the 3' untranslated region (3'-UTR). CRISPRa was applied to validate the putative binding site and an increase in endogenous miR-2861 was found to repress the expression of a reporter that contained the novel miR-2861 binding site. The delivery of miR-2861 to human MSCs enhanced osteoblast differentiation. In the 3'-UTR, the HDAC5 repression was mediated by the miR-2861 binding site, and miR-2861 promoted osteoblast differentiation via the inhibition of HDAC5 through a primate-specific miRNA binding site. Therefore, miRNAmiR-2861 with the CRISPRa methods might be a good biomaterial for osteogenesis augmentation., (© 2024. The Author(s).)

The recently developed CRISPR activator (CRISPRa) system uses a CRISPR-Cas effector-based transcriptional activator to effectively control the expression of target genes without causing DNA damage. However, existing CRISPRa systems based on Cas9/Cas12a necessitate improvement in terms of efficacy and accuracy due to limitations associated with the CRISPR-Cas module itself. To overcome these limitations and effectively and accurately regulate gene expression, we developed an efficient CRISPRa system based on the small CRISPR-Cas effector Candidatus Woesearchaeota Cas12f (CWCas12f). By engineering the CRISPR-Cas module, linking activation domains, and using various combinations of linkers and nuclear localization signal sequences, the optimized eCWCas12f-VPR system enabled effective and target-specific regulation of gene expression compared with that using the existing CRISPRa system. The eCWCas12f-VPR system developed in this study has substantial potential for controlling the transcription of endogenous genes in living organisms and serves as a foundation for future gene therapy and biological research., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Purpose: One of the novel cell sources of cell-based liver regenerative medicine is human chemically-derived hepatic progenitors (hCdHs). We previously established this cell by direct hepatocyte reprogramming with a combination of small molecules (hepatocyte growth factor, A83-01, CHIR99021). However, there have been several issues concerning the cell's stability and maintenance, namely the occurrences of epithelial-mesenchymal transition (EMT) that develop fibrotic phenotypes, resulting in the loss of hepatic progenitor characteristics. These hepatic progenitor attributes are thought to be regulated by SOX9, a transcription factor essential for hepatic progenitor cells and cholangiocytes., Methods: To suppress the fibrotic phenotype and improve our long-term hCdHs culture technology, we utilized the epigenetic modulating drugs DNA methyltransferase inhibitor (5-azacytidine) and histone deacetylase inhibitor (sodium butyrate) that have been reported to suppress and revert hepatic fibrosis. To confirm the essential role of SOX9 to our cell, we used clustered regularly interspaced short palindromic repeats-interference (CRISPRi) to repress the SOX9 expression., Results: The treatment of only 5-azacytidine significantly reduces the fibrosis/mesenchymal marker and EMT-related transcription factor expression level in the early passages. Interestingly, this treatment also increased the hepatic progenitor markers expression, even during the reprogramming phase. Then, we confirmed the essential role of SOX9 by repressing the SOX9 expression with CRISPRi which resulted in the downregulation of several essential hepatic progenitor cell markers., Conclusion: These results highlight the capacity of 5-azacytidine to inhibit EMT-driven hepatic fibrosis and the significance of SOX9 on hepatic progenitor cell stemness properties., Competing Interests: Conflict of Interest: Michael Adisasmita is supported by Hyundai Motor Chung Mong-Koo Global Scholarship. No other potential conflicts of interest relevant to this article were reported., (Copyright © 2024, the Korean Surgical Society.)

The CRISPR-Cas nickase system for genome editing has attracted considerable attention owing to its safety, efficiency, and versatility. Although alternative effectors to Cas9 have the potential to expand the scope of genome editing, their application has not been optimized. Herein, we used an enhanced CRISPR-Cas12a nickase system to induce mutations by targeting genes in a human-derived cell line. The optimized CRISPR-Cas12a nickase system effectively introduced mutations into target genes under a specific directionality and distance between nickases. In particular, the single-mode Cas12a nickase system can induce the target-specific mutations with less DNA double-strand breaks. By inducing mutations in the Thymine-rich target genes in single- or dual-mode, Cas12a nickase compensates the limitations of Cas9 nickase and is expected to contribute to the development of future genome editing technologies., (© 2024. The Author(s).)

Spatial transcriptomics is a newly emerging field that enables high-throughput investigation of the spatial localization of transcripts and related analyses in various applications for biological systems. By transitioning from conventional biological studies to "in situ" biology, spatial transcriptomics can provide transcriptome-scale spatial information. Currently, the ability to simultaneously characterize gene expression profiles of cells and relevant cellular environment is a paradigm shift for biological studies. In this review, recent progress in spatial transcriptomics and its applications in neuroscience and cancer studies are highlighted. Technical aspects of existing technologies and future directions of new developments (as of March 2023), computational analysis of spatial transcriptome data, application notes in neuroscience and cancer studies, and discussions regarding future directions of spatial multi-omics and their expanding roles in biomedical applications are emphasized., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Tissue clearing combined with deep imaging has emerged as a powerful technology to expand classical histological techniques. Current techniques have been optimized for imaging sparsely pigmented organs such as the mammalian brain. In contrast, melanin-rich pigmented tissue, of great interest in the investigation of melanomas, remains challenging. To address this challenge, we have developed a CRISPR-based gene editing approach that is easily incorporated into existing tissue-clearing workflows such the PACT clearing method. We term this method CRISPR-Clear. We demonstrate its applicability to highly melanin-rich B16-derived solid tumors, including one made transgenic for HER2, constituting one of very few syngeneic mouse tumors that can be used in immunocompetent models. We demonstrate the utility in detailed tumor characterization by staining for targeting antibodies and nanoparticles, as well as expressed fluorescent proteins. With CRISPR-Clear we have unprecedented access to optical interrogation in considerable portions of intact melanoma tissue for stained surface markers, expressed fluorescent proteins, of subcellular compartments, and of the vasculature., (© 2023. The Author(s).)

Terahertz (THz) radiation can affect the degree of DNA methylation, the spectral characteristics of which exist in the terahertz region. DNA methylation is an epigenetic modification in which a methyl (CH 3 ) group is attached to cytosine, a nucleobase in human DNA. Appropriately controlled DNA methylation leads to proper regulation of gene expression. However, abnormal gene expression that departs from controlled genetic transcription through aberrant DNA methylation may occur in cancer or other diseases. In this study, we demonstrate the modification of gene expression in cells by THz demethylation using resonant THz radiation. Using an enzyme-linked immunosorbent assay, we observed changes in the degree of global DNA methylation in the SK-MEL-3 melanoma cell line under irradiation with 1.6-THz radiation with limited spectral bandwidth. Resonant THz radiation demethylated living melanoma cells by 19%, with no significant occurrence of apurinic/apyrimidinic sites, and the demethylation ratio was linearly proportional to the power of THz radiation. THz demethylation downregulates FOS, JUN, and CXCL8 genes, which are involved in cancer and apoptosis pathways. Our results show that THz demethylation has the potential to be a gene expression modifier with promising applications in cancer treatment., (© 2023. The Author(s).)

We present a novel genome-wide off-target prediction method named Extru-seq and compare it with cell-based (GUIDE-seq), in vitro (Digenome-seq), and in silico methods using promiscuous guide RNAs with large numbers of valid off-target sites. Extru-seq demonstrates a high validation rate and retention of information about the intracellular environment, both beneficial characteristics of cell-based methods. Extru-seq also shows a low miss rate and could easily be performed in clinically relevant cell types with little optimization, which are major positive features of the in vitro methods. In summary, Extru-seq shows beneficial features of cell-based and in vitro methods., (© 2023. The Author(s).)

Bacterial sphingomyelinases (SMases) hydrolyze sphingomyelin and play an important role in membrane dynamics and the host immune system. While the number of sequenced genomes and metagenomes is increasing, a limited number of experimentally validated SMases have been reported, and the genomic diversity of SMases needs to be elucidated extensively. This study investigated the sequence and structural characteristics of SMases in bacterial genomes and metagenomes. Using previously identified SMases, such as the β-toxin of Staphylococcus aureus , we identified 276 putative SMases and 15 metagenomic SMases by a sequence homology search. Among the predicted metagenomic SMases, six non-redundant metagenomic SMases (M-SMase1-6) were selected for further analysis. The predicted SMases were confirmed to contain highly conserved residues in the central metal-binding site; however, the edge metal-binding site showed high diversity according to the taxon. In addition, protein structure modeling of metagenomic SMases confirmed structural conservation of the central metal-binding site and variance of the edge metal-binding site. From the activity assay on M-SMase2 and M-SMase5, we found that they displayed sphingomyelinase activity compared to Bacillus cereus SMase. This study elucidates a comprehensive genomic characterization of SMases and provides insight into the sequence-structure-activity relationship., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jeon, Kang, Hur and Rho.)

Background: The activation of the telomere maintenance mechanism (TMM) is one of the critical drivers of cancer cell immortality. In gliomas, TERT expression and TERT promoter mutation are considered to reliably indicate telomerase activation, while ATRX mutation and/or loss indicates an alternative lengthening of telomeres (ALT). However, these relationships have not been extensively validated in tumor tissues., Methods: Telomerase repeated amplification protocol (TRAP) and C-circle assays were used to profile and characterize the TMM cross-sectionally (n = 412) and temporally (n = 133) across glioma samples. WES, RNA-seq, and NanoString analyses were performed to identify and validate the genetic characteristics of the TMM groups., Results: We show through the direct measurement of telomerase activity and ALT in a large set of glioma samples that the TMM in glioma cannot be defined solely by the combination of telomerase activity and ALT, regardless of TERT expression, TERT promoter mutation, and ATRX loss. Moreover, we observed that a considerable proportion of gliomas lacked both telomerase activity and ALT. This telomerase activation-negative and ALT negative group exhibited evidence of slow growth potential. By analyzing a set of longitudinal samples from a separate cohort of glioma patients, we discovered that the TMM is not fixed and can change with glioma progression., Conclusions: This study suggests that the TMM is dynamic and reflects the plasticity and oncogenicity of tumor cells. Direct measurement of telomerase enzyme activity and evidence of ALT should be considered when defining TMM. An accurate understanding of the TMM in glioma is expected to provide important information for establishing cancer management strategies., (© 2022. The Author(s).)

Salivary glands that produce and secrete saliva, which is essential for lubrication, digestion, immunity, and oral homeostasis, consist of diverse cells. The long-term maintenance of diverse salivary gland cells in organoids remains problematic. Here, we establish long-term murine and human salivary gland organoid cultures. Murine and human salivary gland organoids express gland-specific genes and proteins of acinar, myoepithelial, and duct cells, and exhibit gland functions when stimulated with neurotransmitters. Furthermore, human salivary gland organoids are established from isolated basal or luminal cells, retaining their characteristics. Single-cell RNA sequencing also indicates that human salivary gland organoids contain heterogeneous cell types and replicate glandular diversity. Our protocol also enables the generation of tumoroid cultures from benign and malignant salivary gland tumor types, in which tumor-specific gene signatures are well-conserved. In this study, we provide an experimental platform for the exploration of precision medicine in the era of tissue regeneration and anticancer treatment., (© 2022. The Author(s).)

Prime editing can induce a desired base substitution, insertion, or deletion in a target gene using reverse transcriptase after nick formation by CRISPR nickase. In this study, we develop a technology that can be used to insert or replace external bases in the target DNA sequence by linking reverse transcriptase to the Francisella novicida Cas9, which is a CRISPR-Cas9 ortholog. Using FnCas9(H969A) nickase, the targeting limitation of existing Streptococcus pyogenes Cas9 nickase [SpCas9(H840A)]-based prime editing is dramatically extended, and accurate prime editing is induced specifically for the target genes in human cell lines., (© 2022. The Author(s).)

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system is composed of a Cas12a effector that acts as a DNA-cleaving endonuclease and a crispr RNA (crRNA) that guides the effector to the target DNA. It is considered a key molecule for inducing target-specific gene editing in various living systems. Here, we improved the efficiency and specificity of the CRISPR-Cas12a system through protein and crRNA engineering. In particular, to optimize the CRISPR-Cas12a system at the molecular level, we used a chimeric DNA-RNA guide chemically similar to crRNA to maximize target sequence specificity. Compared with the wild-type (wt)-Cas12a system, when using enhanced Cas12a system (en-Cas12a), the efficiency and target specificity improved on average by 2.58 and 2.77 times, respectively. In our study, when the chimeric DNA-RNA-guided en-Cas12a effector was used, the gene-editing efficiency and accuracy were simultaneously increased. These findings could contribute to highly accurate genome editing, such as human gene therapy, in the near future., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Glioblastomas (GBM) exhibit intratumoral heterogeneity of various oncogenic evolutional processes. We have successfully isolated and established two distinct cancer cell lines with different morphological and biological characteristics that were derived from the same tissue sample of a GBM. When we compared their genomic and transcriptomic characteristics, each cell line harbored distinct mutation clusters while sharing core driver mutations. Transcriptomic analysis revealed that one cell line was undergoing a mesenchymal transition process, unlike the other cell line. Furthermore, we could identify four tumor samples containing our cell line-like clusters from the publicly available single-cell RNA-seq data, and in a set of paired longitudinal GBM samples, we could confirm three pairs where the recurrent sample was enriched in the genes specific to our cell line undergoing mesenchymal transition. The present study provides direct evidence and a valuable source for investigating the ongoing process of subcellular mesenchymal transition in GBM, which has prognostic and therapeutic implications., (© 2022. The Author(s).)

Despite the important roles of dendritic cells (DCs) in airway allergies, current therapeutic strategies such as drugs, allergen immunotherapy and biologics haven't been targeted at them. In this study, we established a promising DC-based therapeutic approach for the alleviation of allergic rhinitis (AR)-associated allergic reactions, using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated targeted gene disruption. RNA sequencing analysis revealed upregulation of vacuolar protein sorting 37 B (VPS37B) in AR-derived DCs, indicating a novel molecular target. Following antigen presentation, VPS37A and VPS37B enabled endocytosis of the mannose receptor, which recognizes the house dust mite (HDM) allergen Der p 1. DCs with targeted disruption of VPS37A/B alleviated Th2 cytokine production when co-cultured in vitro with allogeneic naïve CD4 + T cell from patients with AR. Furthermore, nasal administration of Vps37a/b-disrupted bone marrow DCs to a mouse model of AR resulted in strongly reduced AR-related symptoms. Thus, this novel modality using genetically engineered DCs can provide an effective therapeutic and preventative strategy for allergic diseases., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A turn-on fluorescent nanoprobe (named AAP-1), based on an aggregation-induced emission luminogen (AIEgen), is disclosed for the detection of adenosine triphosphate (ATP), which is an essential element in the biological system. Organic fluorophore (named TPE-TA) consists of tetraphenylethylene (TPE, sensing and signaling moiety) and mono-triamine (TA, sensing moiety), and it forms an aggregated form in aqueous media as a nanoprobe AAP-1. The nanoprobe AAP-1 has multiple electrostatic interactions as well as hydrophobic interactions with ATP, and it displays superior selectivity toward ATP, reliable sensitivity, with a detection limit around 0.275 ppb, and fast responsive (signal within 10 s). Such a fluorescent probe to monitor ATP has been actively pursued throughout fundamental and translational research areas. In vitro assay and a successful cellular ATP imaging application was demonstrated in cancer cells and embryonic stem cells. We expect that our work warrants further ATP-related studies throughout a variety of fields., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interestsThe authors declare the following competing financial interest(s): the authors are listed as inventors on a pending patent application related to the technology described in this work., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a family of DNA sequences originally discovered as a type of acquired immunity in prokaryotes such as bacteria and archaea. In many CRISPR systems, the functional ribonucleoproteins (RNPs) are composed of CRISPR protein and guide RNAs. They selectively bind and cleave specific target DNAs or RNAs, based on sequences complementary to the guide RNA. The specific targeted cleavage of the nucleic acids by CRISPR has been broadly utilized in genome editing methods. In the process of genome editing of eukaryotic cells, CRISPRmediated DNA double-strand breaks (DSB) at specific genomic loci activate the endogenous DNA repair systems and induce mutations at the target sites with high efficiencies. Two of the major endogenous DNA repair machineries are non-homologous end joining (NHEJ) and homology-directed repair (HDR). In case of DSB, the two repair pathways operate in competition, resulting in several possible outcomes including deletions, insertions, and substitutions. Due to the inherent stochasticity of DSB-based genome editing methods, it was difficult to achieve defined single-base changes without unanticipated random mutation patterns. In order to overcome the heterogeneity in DSB-mediated genome editing, novel methods have been developed to incorporate precise single-base level changes without inducing DSB. The approaches utilized catalytically compromised CRISPR in conjunction with base-modifying enzymes and DNA polymerases, to accomplish highly efficient and precise genome editing of single and multiple bases. In this review, we introduce some of the advances in single-base level CRISPR genome editing methods and their applications. [BMB Reports 2021; 54(2): 98-105].

Lentiviruses have been widely used as a means of transferring exogenous DNAs into human cells to treat various genetic diseases. Lentiviral vectors are fundamentally integrated into the host genome, but their integration sites are generally unpredictable, which may increase the uncertainty for their use in therapeutics. To determine the viral integration sites in the host genome, several PCR-based methods have been developed. However, the sensitivities of the PCR-based methods are highly dependent on the primer sequences, and optimized primer design is required for individual target sites. In order to address this issue, we developed an alternative method for genome-wide mapping of viral insertion sites, named CReVIS-seq (CRISPR-enhanced Viral Integration Site Sequencing). The method is based on the sequential steps: fragmentation of genomic DNAs, in vitro circularization, cleavage of target sequence in a CRISPR guide RNA-specific manner, high-throughput sequencing of the linearized DNA fragments in an unbiased manner, and identification of viral insertion sites via sequence analysis. By design, CReVIS-seq is not affected by biases that could be introduced during the target enrichment step via PCR amplification using site specific primers. Furthermore, we found that multiplexed CReVIS-seq, using collections of different single-guide RNAs (sgRNAs), enables simultaneous identification of multiple target sites and structural variations (i.e., circularized viral genome), in both single cell clones and heterogeneous cell populations., Competing Interests: H.S.K. and J.K.H. are co-inventors on a patent application covering the CReVIS-seq method described in this manuscript. The remaining authors declare no competing interests., (© 2020 The Authors.)

Multidrug-resistant (MDR) bacteria have become a severe problem for public health. Developing new antibiotics for MDR bacteria is difficult, from inception to the clinically approved stage. Here, we have used a new approach, modification of an antibiotic, ciprofloxacin (CFX), with triphenylphosphonium (TPP, PPh 3 ) moiety via ester- (CFX-ester-PPh 3 ) and amide-coupling (CFX-amide-PPh 3 ) to target bacterial membranes. In this study, we have evaluated the antibacterial activities of CFX and its derivatives against 16 species of bacteria, including MDR bacteria, using minimum inhibitory concentration (MIC) assay, morphological monitoring, and expression of resistance-related genes. TPP-conjugated CFX, CFX-ester-PPh 3 , and CFX-amide-PPh 3 showed significantly improved antibacterial activity against Gram-positive bacteria, Staphylococcus aureus , including MDR S. aureus (methicillin-resistant S. aureus (MRSA)) strains. The MRSA ST5 5016 strain showed high antibacterial activity, with MIC values of 11.12 µg/mL for CFX-ester-PPh 3 and 2.78 µg/mL for CFX-amide-PPh 3 . The CFX derivatives inhibited biofilm formation in MRSA by more than 74.9% of CFX-amide-PPh 3 . In the sub-MIC, CFX derivatives induced significant morphological changes in MRSA, including irregular deformation and membrane disruption, accompanied by a decrease in the level of resistance-related gene expression. With these promising results, this method is very likely to combat MDR bacteria through a simple TPP moiety modification of known antibiotics, which can be readily prepared at clinical sites.

The CRISPR-Cas9 system is widely used for target-specific genome engineering. CRISPR-Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

CRISPR effectors, which comprise a CRISPR-Cas protein and a guide (g)RNA derived from the bacterial immune system, are widely used for target-specific genome editing. When the gRNA recognizes genomic loci with sequences that are similar to the target, deleterious mutations can occur. Off-target mutations with a frequency below 0.5% remain mostly undetected by current genome-wide off-target detection techniques. Here we report a method to effectively detect extremely small amounts of mutated DNA based on predicted off-target-specific amplification. In this study, we used various genome editors to induce intracellular genome mutations, and the CRISPR amplification method detected off-target mutations at a significantly higher rate (1.6~984 fold increase) than an existing targeted amplicon sequencing method. In the near future, CRISPR amplification in combination with genome-wide off-target detection methods will allow detection of genome editor-induced off-target mutations with high sensitivity and in a non-biased manner.

ChIP-seq is one of the core experimental resources available to understand genome-wide epigenetic interactions and identify the functional elements associated with diseases. The analysis of ChIP-seq data is important but poses a difficult computational challenge, due to the presence of irregular noise and bias on various levels. Although many peak-calling methods have been developed, the current computational tools still require, in some cases, human manual inspection using data visualization. However, the huge volumes of ChIP-seq data make it almost impossible for human researchers to manually uncover all the peaks. Recently developed convolutional neural networks (CNN), which are capable of achieving human-like classification accuracy, can be applied to this challenging problem. In this study, we design a novel supervised learning approach for identifying ChIP-seq peaks using CNNs, and integrate it into a software pipeline called CNN-Peaks. We use data labeled by human researchers who annotate the presence or absence of peaks in some genomic segments, as training data for our model. The trained model is then applied to predict peaks in previously unseen genomic segments from multiple ChIP-seq datasets including benchmark datasets commonly used for validation of peak calling methods. We observe a performance superior to that of previous methods.

Two of the most critical factors for the survival of glioblastoma (GBM) patients are precision diagnosis and the tracking of treatment progress. At the moment, various sophisticated and specific diagnostic procedures are being used, but there are relatively few simple diagnosis methods. This work introduces a sensing probe based on a turn-on type fluorescence response that can measure the cysteine (Cys) level, which is recognized as a new biomarker of GBM, in human-derived cells and within on-site human clinical biopsy samples. The Cys-initiated chemical reactions of the probe cause a significant fluorescence response with high selectivity, high sensitivity, a fast response time, and a two-photon excitable excitation pathway, which allows the imaging of GBM in both mouse models and human tissue samples. The probe can distinguish the GBM cells and disease sites in clinical samples from individual patients. Besides, the probe has no short or long-term toxicity and immune response. The present findings hold promise for application of the probe to a relatively simple and straightforward following of GBM at clinical sites., (This journal is © The Royal Society of Chemistry 2020.)

Articulated structures of naphthalene-based donor (D)-acceptor (A) type dipolar dye and aggregation-induced emission luminogen (AIEgen) based on tetraphenylethylene (TPE) were synthesized, and their photophysical properties were analyzed for the first time. There are many fluorophore backbones, which have dipolar structure and AIEgen. However, there has been neither property analysis nor research that closely articulates DA and AIE through non-conjugation linker. We have therefore prepared two representative fluorophores; DA-AIE series (DA-AIE-M and DA-AIE-D), and characterized their UV/vis absorption and emission properties with quantum chemical calculations. In addition, we utilized the unique photophysical properties of DA-AIE-D for monitoring a trace of dimethyl sulfoxide (DMSO) in aqueous media, including real water samples.

Clustered regularly interspaced short palindromic repeats, or CRISPR, has been widely accepted as a versatile genome editing tool with significant potential for medical application. Reliable allele specificity is one of the most critical elements for successful application of this technology to develop high-precision therapeutics and diagnostics. CRISPR-based genome editing tools achieve high-fidelity distinction of single-base differences in target genomic loci by structural identification of CRISPR-associated (Cas) proteins and sequences of the guide RNAs. In this review, we describe the structural features of ribonucleoprotein complex formation by CRISPR proteins and guide RNAs that eventually recognize target DNA sequences. This structural understanding provides the basis for the recent applications of enhanced single-base precision genome editing technologies for effective distinction of specific alleles., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A latent turn-on fluorescent probe for the detection of malononitrile (NCCH 2 CN), a precursor of hydrogen cyanide (HCN) in the mammalian tissue metabolism, is developed based on reaction-based fluorophore generation for the first time. Malononitrile is utilized within a wide spectrum of academic and industrial applications, and it is a key reagent to make o-chlorobenzylidene malononitrile (CS gas; tear gas), which is used for riot control. Due to its extensive use as well as potential health risks and the environmental pollution, malononitrile monitoring method has been required. In this paper, we discovered that our key sensing platform, 6-(dimethylamino)-3-hydroxy-2-naphthaldehyde (named Mal-P1), responds sensitively and selectively towards malononitrile. The Knoevenagel condensation induced benzo [g]coumarin formation of Mal-P1 with malononitrile showed significant fluorescence turn-on response. In addition, Mal-P1 showed the malononitrile sensing ability in environmental samples (real water, CS gas) and imaging ability in biological sample (HeLa cell line) using fluorescence microscopy with low cytotoxicity. The successful demonstrations will facilitate further applications in a variety of fields., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The CRISPR systems provide powerful genome-editing tools for wide applications in biological and medical research fields. However, the safety issue due to off-target effects of CRISPR has been one of the major hindrances of its application to regenerative medicine. The conventional CRISPR system has the intrinsic danger of inducing unpredictable mutations at non-targeted genomic loci via erroneous double-strand DNA breaks (DSBs). In this study, we demonstrate a safety-enhanced application of a recently discovered CRISPR-Cpf1 for targeted gene activation, without DNA double-strand break, to facilitate osteogenic differentiation of human umbilical-cord-derived mesenchymal stem cells (UC-MSCs). To this end, we developed a catalytically inactive AsCpf1 fused to tripartite transcription activator domain (dAsCpf1-VPR) that can induce upregulation of targeted gene expression in mammalian cells. We observed that the CRISPR-dAsCpf1-VPR activator can be applied to enhance the osteogenic differentiation of human UC-MSCs, via increasing the expression level of endogenous BMP4 gene. The results suggested that the CRISPR-Cpf1 activator provides versatile methods applicable for bone regeneration and regenerative medicine., (© 2020 The Authors.)

In CRISPR genome editing, CRISPR proteins form ribonucleoprotein complexes with guide RNAs to bind and cleave the target DNAs with complete sequence complementarity. CRISPR genome editing has a high potential for use in precision gene therapy for various diseases, including cancer and genetic disorders, which are caused by DNA mutations within the genome. However, several studies have shown that targeting the DNA via sequence complementarity is imperfect and subject to unintended genome editing of other genomic loci with similar sequences. These off-target problems pose critical safety issues in the therapeutic applications of CRISPR technology, with particular concerns in terms of the genome editing of pathogenic point mutations, where non-mutant alleles can become an off-target with only a one-base difference. In this study, we sought to assess a novel CRISPR genome editing technique that has been proposed to achieve a high specificity by positioning the mismatches within the protospacer adjacent motif (PAM) sequence. To this end, we compared the genome editing specificities of the PAM-based and conventional methods on an oncogenic single-base mutation in the endothelial growth factor receptor (EGFR). The results indicated that the PAM-based method provided a significantly increased genome editing specificity for pathogenic mutant alleles with single-base precision.

Ligamentum flavum hypertrophy (LFH) is the most important component of lumbar spinal canal stenosis. Although the pathophysiology of LFH has been extensively studied, no method has been proposed to prevent or treat it. Since the transforming growth factor-β (TGF-β) pathway is known to be critical in LFH pathology, we investigated whether LFH could be prevented by blocking or modulating the TGF-β mechanism. Human LF cells were used for the experiments. First, we created TGF-β receptor 1 (TGFBR1) knock out (KO) cells with CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 biotechnology and treated them with TGF-β1 to determine the effects of blocking the TGF-β pathway. Subsequently, we studied the effect of CCN5, which has recently been proposed to modulate the TGF-β pathway. To assess the predisposition toward fibrosis, α-smooth muscle actin (αSMA), fibronectin, collagen-1, collagen-3, and CCN2 were evaluated with quantitative real-time polymerase chain reaction, western blotting, and immunocytochemistry. The TGFBR1 KO LF cells were successfully constructed with high KO efficiency. In wild-type (WT) cells, treatment with TGF-β1 resulted in the overexpression of the messenger RNA (mRNA) of fibrosis-related factors. However, in KO cells, the responses to TGF-β1 stimulation were significantly lower. In addition, CCN5 and TGF-β1 co-treatment caused a notable reduction in mRNA expression levels compared with TGF-β1 stimulation only. The αSMA protein expression increased with TGF-β1 but decreased with CCN5 treatment. TGF-β1 induced LF cell transdifferentiation from fibroblasts to myofibroblasts. However, this cell transition dramatically decreased in the presence of CCN5. In conclusion, CCN5 could prevent LFH by modulating the TGF-β pathway. © 2019 The Authors. Journal of Orthopaedic Research ® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:2634-2644, 2019., (© 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society.)

A transient cytosolic delivery system for accurate Cas9 ribonucleoprotein is a key factor for target specificity of the CRIPSR/Cas9 toolkit. Owing to the large size of the Cas9 protein and a long negative strand RNA, the development of the delivery system is still a major challenge. Here, a size-controlled lipopeptide-based nanosome system is reported, derived from the blood-brain barrier-permeable dNP2 peptide which is capable of delivering a hyperaccurate Cas9 ribonucleoprotein complex (HypaRNP) into human cells for gene editing. Each nanosome is capable of encapsulating and delivering ≈2 HypaRNP molecules into the cytoplasm, followed by nuclear localization at 4 h post-treatment without significant cytotoxicity. The HypaRNP thus efficiently enacts endogenous eGFP silencing and editing in human embryonic kidney cells (up to 27.6%) and glioblastoma (up to 19.7% frequency of modification). The lipopeptide-based nanosome system shows superior delivery efficiency, high controllability, and simplicity, thus providing biocompatibility and versatile platform approach for CRISPR-mediated transient gene editing applications., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Adenine base editors comprise an adenosine deaminase, evolved in vitro, and a Cas9 nickase. Here, we show that in addition to converting adenine to guanine, adenine base editors also convert cytosine to guanine or thymine in a narrow editing window (positions 5-7) and in a confined TC*N sequence context. Adenine base editor-induced cytosine substitutions occur independently of adenosine conversions with an efficiency of up to 11.2% and reduce the number of suitable targeting sites for high-specificity base editing.

Background: The CRISPR-Cas systems have emerged as a robust genome editing tool useful in various fields of research. With the discovery and development of the orthologous CRISPR-Cas systems, their genome editing efficiency have improved., Objective: In this review, we aim to present the recent developments and applications of the CRISPR-Cas systems., Methods: First, we introduce how the advancements of CRISPR technology enabled genome editing to single base precision. Then, we discuss the CRISPR based methods for targeted transcriptional regulation, epigenome editing, and RNA editing. Finally, we review the CRISPR delivery systems highlighting recent attempts to integrate nanotechnology to develop novel CRISPR delivery modalities., Conclusion: Here, we review the recent trends in CRISPR-based biotechnologies, encompassing genome editing, epigenome regulation and direct RNA targeting and provide an overview of methods employed for CRISPR delivery with an emphasis on the most recent nanotechnology-based delivery strategies. We anticipate that the development of CRISPR based technology will continue to explore novel methods.

Patient-specific human-induced pluripotent stem cells (hiPSCs) hold great promise for the modelling of genetic disorders. However, these cells display wide intra- and interindividual variations in gene expression, which makes distinguishing true-positive and false-positive phenotypes challenging. Data from hiPSC phenotypes and human embryonic stem cells (hESCs) harbouring the same disease mutation are also lacking. Here, we report a comparison of the molecular, cellular and functional characteristics of three congruent patient-specific cell types-hiPSCs, hESCs and direct-lineage-converted cells-derived from currently available differentiation and direct-reprogramming technologies for use in the modelling of Charcot-Marie-Tooth 1A, a human genetic Schwann-cell disorder featuring a 1.4 Mb chromosomal duplication. We find that the chemokines C-X-C motif ligand chemokine-1 (CXCL1) and macrophage chemoattractant protein-1 (MCP1) are commonly upregulated in all three congruent models and in clinical patient samples. The development of congruent models of a single genetic disease using somatic cells from a common patient will facilitate the search for convergent phenotypes.

Background and Aims: Our study aimed to investigate the association between high-density lipoprotein cholesterol (HDL-C) and all-cause and cause-specific mortality in Korean adults., Methods: A total of 365,457 participants aged ≥40 years were selected from the Korean National Health Insurance Service-National Sample Cohort from 2009 to 2015. HDL-C level was categorized into <1.0, 1.0-1.19, 1.2-1.39, 1.4-1.59, 1.6-1.79 (reference), 1.8-1.99, 2.0-2.19 and ≥ 2.20 mmol/L. Cox proportional hazard models were used to examine the association between HDL-C level and mortality risk., Results: In a median 3.5-year follow-up period, 9,350 participants (2.6%) died. Men with HDL-C level of 1.6-1.79 mmol/L and women with HDL-C level of 1.4-1.59 mmol/L had the lowest age-standardized mortality rates for all-cause death. However, for cardiovascular death, men with HDL-C level ≥2.20 mmol/L and women with HDL-C level of 1.8-1.99 mmol/L showed the lowest mortality rate. After adjusting for multiple covariates, the hazard ratios for all-cause and cancer deaths showed a U-shaped relationship with HDL-C level for both sexes. However, there were heterogenetic associations between HDL-C level and mortality risk of subtypes of cardiovascular disease by sex. For other causes of death except for cardiovascular and cancer death, elevated mortality risk was mainly due to external causes (ICD-10 code, S00-T98)., Conclusions: In South Korea, very high HDL-C level was associated with increased risk of all-cause death. However, the increased all-cause mortality risk in people with very high HDL-C level was partly due to mortality risk from external causes., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

A keloid is a type of unusually raised scar. Unlike other raised scars, keloids form larger sizes than the wound site due to overgrowth, generally related to various biological factors. To date, only a few diagnostic and therapeutic methods for keloids have been reported. The high recurrence rates and undesirable side effects of keloids, at the end stage, encourage the invention of novel diagnostic tools, in order to cure keloids at an earlier stage. In this review, we summarize the general information about keloid diagnosis, keloid biomarkers, and recently reported fluorescent probes that can sense the key biomarkers of keloids. The focused description of fluorescent probes for keloid biomarkers and the author's perspective give useful insights in order to design the next-generation diagnostic sensing system for keloids.

Porous silicon nanoparticles (pSiNPs) have been utilized within a wide spectrum of biological studies, as well as in chemistry, chemical biology, and biomedical fields. Recently, pSiNPs have been constantly coming under the spotlight, mostly in biomedical applications, due to their advantages, such as controlled-release drug delivery in vivo by hydrolysis-induced degradation, self-reporting property through long life-time photoluminescence, high loading efficiency of substrate into pore, and the homing to specific cells/organ/bacteria by surface functionalization. However, the systematic degradation rate analysis of surface-functionalized pSiNPs in different biological media has not been conducted yet. In this paper, we prepared four different surface-functionalized pSiNPs samples and analyzed the degradation rate in six different media (DI H₂O (deionized water), PBS (phosphate-buffered saline), HS (human serum), DMEM (Dulbecco's modified Eagle's medium), LB (lysogeny broth), and BHI (brain heart infusion)). The obtained results will now contribute to understanding the correlation between surface functionalization in the pSiNPs and the degradation rate in different biological media. The characterized data with the author's suggestions will provide useful insights in designing the new pSiNPs formulation for biomedical applications.

The discovery and mechanistic understanding of target-specific genome engineering technologies has led to extremely effective and specific genome editing in higher organisms. Target-specific genetic modification technology is expected to have a leading position in future gene therapy development, and has a ripple effect on various basic and applied studies. However, several problems remain and hinder efficient and specific editing of target genomic loci. The issues are particularly critical in precise targeted insertion of external DNA sequences into genomes. Here, we discuss some recent efforts to overcome such problems and present a perspective of future genome editing technologies.

Purpose: Majority of the previous studies compared lumbar spinal stenosis (LSS) and lumbar disc herniation (LDH) patients for analyses of LFH. However, the separation of normal/hypertrophied LF has often been ambiguous and the severity of hypertrophic activity differed. Here, we present a novel analysis scheme for LFH in which myofibroblast is proposed as a major etiological factor for LFH study., Methods: Seventy-one LF patient tissue samples were used for this study. Initially, mRNA levels of the samples were assessed by qRT-PCR: angiopoietin-like protein-2 (ANGPTL2), transforming growth factor-beta1 (TGF-β1), vascular endothelial growth factor (VEGF), interleukin-6, collagen-1, 3, 4, 5, and 11, and elastin. Myofibroblasts were detected by immune stain using α-smooth muscle actin (αSMA) as a marker. To study the myofibroblast in TGF-β pathway, LF tissues were analyzed for protein levels of αSMA/TGF-β1 by Western blot. In addition, from LF cells cultured with exogenous TGF-β1 conditioned medium, expression of αSMA/collagen-1 was assessed and the cell morphology was identified., Results: The comparative analysis of mRNA expression levels (LSS vs LDH) failed to show significant differences in TGF-β1 (p = 0.08); however, we found a significant positive correlation among ANGPTL2, VEGF, TGF-β1, and collagen-1 and 3, which represent common trends in hypertrophic activity (p < 0.05). We detected myofibroblast in the patient samples by αSMA staining, and the protein levels of αSMA were positively correlated with TGF-β1. In LF cell culture, exogenous TGF-β1 upregulated αSMA/collagen-1 mRNA levels and facilitated trans-differentiation to myofibroblast., Conclusions: We conclude that the transition of fibroblast to myofibroblasts via TGF-β pathway is a key linker between inflammation and fibrosis in LFH mechanism.

Urinary tract infections (UTIs) are clinically important problems that lead to serious morbidity and mortality, and indwelling urinary catheters are a major factor of UTIs. In this study, we applied clustered regularly interspaced short palindromic repeats (CRISPR) genome editing to generate ΔluxS mutant strains from clinical isolates of Escherichiacoli SE15, which is one of major pathogens and can cause colonization and biofilm formation in the catheter. A major regulatory pathway of such biofilm formation on medical devices is the quorum sensing mechanism via small molecule autoinducer-2 synthesized by LuxS enzyme. Here, we used the CRISPR-Cas9 system for precise deletion of luxS gene in clinical isolate E. coli SE15. To this end, we constructed a donor DNA for homologous recombination to delete 93 bases in the chromosomal target (luxS) and observed the success rate of luxS deletion to be 22.7 %. We conducted biofilm assay to observe decreased biofilm formation in the E. coil SE15 ΔluxS mutants compared to wild-type E. coil SE15. Quantitative real-time PCR analysis of E. coil SE15 ΔluxS mutants showed that the expression of luxS was below detection level. We also observed that the relative mRNA levels of biofilm-formation-related genes, such as mqsR, pgaBC and csgEF, were significantly decreased in E. coil SE15 ΔluxS mutants compared to wild-type. We conclude that genome editing by CRISPR-Cas9 system is an effective tool to dissect the molecular mechanism of biofilm formation in medically important strains, and the study may serve as a basis for developing novel medical intervention against UTIs caused by biofilm.

Programmable clustered regularly interspaced short palindromic repeats (CRISPR) Cpf1 endonucleases are single-RNA-guided (crRNA) enzymes that recognize thymidine-rich protospacer-adjacent motif (PAM) sequences and produce cohesive double-stranded breaks (DSBs). Genome editing with CRISPR-Cpf1 endonucleases could provide an alternative to CRISPR-Cas9 endonucleases, but the determinants of targeting specificity are not well understood. Using mismatched crRNAs we found that Cpf1 could tolerate single or double mismatches in the 3' PAM-distal region, but not in the 5' PAM-proximal region. Genome-wide analysis of cleavage sites in vitro for eight Cpf1 nucleases using Digenome-seq revealed that there were 6 (LbCpf1) and 12 (AsCpf1) cleavage sites per crRNA in the human genome, fewer than are present for Cas9 nucleases (>90). Most Cpf1 off-target cleavage sites did not produce mutations in cells. We found mismatches in either the 3' PAM-distal region or in the PAM sequence of 12 off-target sites that were validated in vivo. Off-target effects were completely abrogated by using preassembled, recombinant Cpf1 ribonucleoproteins.

The THO/TREX complex consists of several conserved subunits and is required for mRNA export. In metazoans, THO/TREX binds a subset of mRNAs during RNA splicing, and facilitates their nuclear export. How THO/TREX selects RNA targets is, however, incompletely understood. In our recent study, we reported that THO is loaded onto Piwi-interacting RNA (piRNA) precursor transcripts independent of splicing, and facilitates convergent transcription in Drosophila ovary. The precursors are later processed into mature piRNAs, small noncoding RNAs that silence transposable elements (TEs). We observed that piRNAs originating from dual-strand clusters, where precursors are transcribed from both strands, were specifically affected by THO mutation. Analysis of THO-bound RNAs showed enrichment of dual-strand cluster transcripts. Interestingly, THO loading onto piRNA precursors was dependent on Cutoff (Cuff), which comprises the Rhino-Deadlock-Cutoff (RDC) complex that is recruited to dual-strand clusters by recognizing H3K9me3 and licenses convergent transcription from the cluster. We also found that THO mutation affected transcription from dualstrand clusters. Therefore, we concluded that THO/TREX is recruited to dual-strand piRNA clusters, independent of splicing events, via multi-protein interactions with chromatin structure. Then, it facilitates transcription likely by suppressing premature termination to ensure adequate expression of piRNA precursors. [BMB Reports 2016; 49(7): 355-356].