Hypoxia, oxidative stress, and the interplay of HIFs and NRF2 signaling in cancer.

Oxygen is crucial for life and acts as the final electron acceptor in mitochondrial energy production. Cells adapt to varying oxygen levels through intricate response systems. Hypoxia-inducible factors (HIFs), including HIF-1α and HIF-2α, orchestrate the cellular hypoxic response, activating genes to increase the oxygen supply and reduce expenditure. Under conditions of excess oxygen and resulting oxidative stress, nuclear factor erythroid 2-related factor 2 (NRF2) activates hundreds of genes for oxidant removal and adaptive cell survival. Hypoxia and oxidative stress are core hallmarks of solid tumors and activated HIFs and NRF2 play pivotal roles in tumor growth and progression. The complex interplay between hypoxia and oxidative stress within the tumor microenvironment adds another layer of intricacy to the HIF and NRF2 signaling systems. This review aimed to elucidate the dynamic changes and functions of the HIF and NRF2 signaling pathways in response to conditions of hypoxia and oxidative stress, emphasizing their implications within the tumor milieu. Additionally, this review explored the elaborate interplay between HIFs and NRF2, providing insights into the significance of these interactions for the development of novel cancer treatment strategies.

CRISPR-clear imaging of melanin-rich B16-derived solid tumors.

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.

CReVIS-Seq: A highly accurate and multiplexable method for genome-wide mapping of lentiviral integration sites.

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.

Ectopic transient overexpression of OCT-4 facilitates BMP4-induced osteogenic transdifferentiation of human umbilical vein endothelial cells.

Limitation in cell sources for autologous cell therapy has been a recent focus in stem cell therapy and tissue engineering. Among various research advances, direct conversion, or transdifferentiation, is a notable and feasible strategy for the generation and acquirement of wanted cell source. So far, utilizing cell transdifferentiation technology in tissue engineering was mainly restricted at achieving single wanted cell type from diverse cell types with high efficiency. However, regeneration of a complete tissue always requires multiple cell types which poses an intrinsic complexity. In this study, enhanced osteogenic differentiation was achieved by transient ectopic expression of octamer-binding transcription factor 4 (OCT-4) gene followed by bone morphogenetic protein 4 treatment on human umbilical vein endothelial cells. OCT-4 transfection and bone morphogenetic protein 4 treatment resulted in enhanced expression of osteogenic markers such as core-binding factor alpha 1, alkaline phosphatase, and collagen 1 compared with bone morphogenetic protein 4 treatment alone. Furthermore, we employed gelatin-heparin cryogel in cranial defect model for in vivo bone formation. Micro-computed tomography and histological analysis of in vivo samples showed that OCT-4 transfection followed by bone morphogenetic protein 4 treatment resulted in efficient transdifferentiation of endothelial cells to osteogenic cells. These results suggest that the combination of OCT-4 and bone morphogenetic protein 4 on endothelial cells would be a reliable multicellular transdifferentiation model which could be applied for bone tissue engineering.

CRISPR-Cpf1 Activation of Endogenous BMP4 Gene for Osteogenic Differentiation of Umbilical-Cord-Derived Mesenchymal Stem Cells.

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.

Specificity Assessment of CRISPR Genome Editing of Oncogenic EGFR Point Mutation with Single-Base Differences.

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.

Lipopeptide-Based Nanosome-Mediated Delivery of Hyperaccurate CRISPR/Cas9 Ribonucleoprotein for Gene Editing.

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.

CCN5 Reduces Ligamentum Flavum Hypertrophy by Modulating the TGF-ß Pathway.

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-ß (TGF-ß) pathway is known to be critical in LFH pathology, we investigated whether LFH could be prevented by blocking or modulating the TGF-ß mechanism. Human LF cells were used for the experiments. First, we created TGF-ß receptor 1 (TGFBR1) knock out (KO) cells with CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 biotechnology and treated them with TGF-ß1 to determine the effects of blocking the TGF-ß pathway. Subsequently, we studied the effect of CCN5, which has recently been proposed to modulate the TGF-ß 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-ß1 resulted in the overexpression of the messenger RNA (mRNA) of fibrosis-related factors. However, in KO cells, the responses to TGF-ß1 stimulation were significantly lower. In addition, CCN5 and TGF-ß1 co-treatment caused a notable reduction in mRNA expression levels compared with TGF-ß1 stimulation only. The αSMA protein expression increased with TGF-ß1 but decreased with CCN5 treatment. TGF-ß1 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-ß 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.

Recent trends in CRISPR-Cas system: genome, epigenome, and transcriptome editing and CRISPR delivery systems.

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.

CRISPR/Cas9 searches for a protospacer adjacent motif by lateral diffusion.

The Streptococcus pyogenes CRISPR/Cas9 (SpCas9) nuclease has been widely applied in genetic engineering. Despite its importance in genome editing, aspects of the precise molecular mechanism of Cas9 activity remain ambiguous. In particular, because of the lack of a method with high spatio-temporal resolution, transient interactions between Cas9 and DNA could not be reliably investigated. It therefore remains controversial how Cas9 searches for protospacer adjacent motif (PAM) sequences. We have developed single-molecule Förster resonance energy transfer (smFRET) assays to monitor transient interactions of Cas9 and DNA in real time. Our study shows that Cas9 interacts with the PAM sequence weakly, yet probing neighboring sequences via facilitated diffusion. This dynamic mode of interactions leads to translocation of Cas9 to another PAM nearby and consequently an on-target sequence. We propose a model in which lateral diffusion competes with three-dimensional diffusion and thus is involved in PAM finding and consequently on-target binding. Our results imply that the neighboring sequences can be very important when choosing a target in genetic engineering applications.

Rescue of high-specificity Cas9 variants using sgRNAs with matched 5' nucleotides.

We report that engineered Cas9 variants with improved specificity-eCas9-1.1 and Cas9-HF1-are often poorly active in human cells, when complexed with single guide RNAs (sgRNAs) with a mismatch at the 5' terminus, relative to target DNA sequences. Because the nucleotide at the 5' end of sgRNAs, expressed under the control of the commonly-used U6 promoter, is fixed to a guanine, these attenuated Cas9 variants are not useful at many target sites. By using sgRNAs with matched 5' nucleotides, produced by linking them to a self-cleaving ribozyme, the editing activity of Cas9 variants can be rescued without sacrificing high specificity.

Myofibroblast in the ligamentum flavum hypertrophic activity.

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-ß1), 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-ß pathway, LF tissues were analyzed for protein levels of αSMA/TGF-ß1 by Western blot. In addition, from LF cells cultured with exogenous TGF-ß1 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-ß1 (p = 0.08); however, we found a significant positive correlation among ANGPTL2, VEGF, TGF-ß1, 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-ß1. In LF cell culture, exogenous TGF-ß1 upregulated αSMA/collagen-1 mRNA levels and facilitated trans-differentiation to myofibroblast. CONCLUSIONS: We conclude that the transition of fibroblast to myofibroblasts via TGF-ß pathway is a key linker between inflammation and fibrosis in LFH mechanism.