Clustered Regularly Interspaced Short Palindromic repeats/Cas9 Research Articles

Progress in Research and Prospects for Application of Precision Gene-Editing Technology Based on CRISPR–Cas9 in the Genetic Improvement of Sheep and Goats

Due to recent innovations in gene editing technology, great progress has been made in livestock breeding, with researchers rearing gene-edited pigs, cattle, sheep, and other livestock. Gene-editing technology involves knocking in, knocking out, deleting, inhibiting, activating, or replacing specific bases of DNA or RNA sequences at the genome level for accurate modification, and such processes can edit genes at a fixed point without needing DNA templates. In recent years, although clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system-mediated gene-editing technology has been widely used in research into the genetic breeding of animals, the system’s efficiency at inserting foreign genes is not high enough, and there are certain off-target effects; thus, it is not appropriate for use in the genome editing of large livestock such as cashmere goats. In this study, the development status, associated challenges, application prospects, and future prospects of CRISPR/Cas9-mediated precision gene-editing technology for use in livestock breeding were reviewed to provide a theoretical reference for livestock gene function analysis, genetic improvement, and livestock breeding that account for characteristics of local economies.

Consumer acceptance and valuation of quality‐improved food products derived by genome editing technology. A case study of rice in Vietnam

AbstractUnlike genetic modification, genome editing (GE) technologies can be used to yield transgene‐free outcomes, which is an important aspect in promoting consumer acceptance of GE foods. In addition, with the advent of the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system, which is known to be exceptional among genome editing tools, GE has numerous potential applications in plant breeding technology to create diverse desirable traits, especially consumer‐targeted traits such as improved product quality and nutrition. It is expected that the GE foods market will overtake that of genetically modified (GM) foods. Although few GE products have been introduced to the market, some studies have already evaluated consumer acceptance and valuation of GE foods in comparison with GM and conventional foods. However, these studies mainly focused on traits relevant to cultivation efficiency and ignored consumer preferences for desirable traits. Further, it has been shown that consumers evaluate GE foods somewhat higher than GM foods; yet, as observed for GM foods, consumers expect a discounted price for GE foods. GE application for consumer‐targeted traits could, however, have a potentially positive effect on consumer acceptance. This study was conducted to evaluate consumer acceptance and valuation of quality‐improved consumer‐targeted GE products. We defined the determinants and estimated the willingness to pay a price premium for GE rice compared to GM and conventional rice by using the double‐bounded contingent valuation method under different information treatments. The survey was conducted in Vietnam, where consumers have not been exposed to information regarding GE via social media that could lead to a biased perspective. This context is ideal for investigating the effect of information provision during the introductory stage of GE products to the market. Our main findings suggest that consumers will widely accept quality‐improved GE foods targeted at consumer preferences, as well as the positive influence of in‐depth information provision on potential consumer acceptance. [EconLit Citations: Q10: Agriculture: General].

The History, Use, and Challenges of Therapeutic Somatic Cell and Germline Gene Editing

ABSTRACT Gene editing is a technology that has been rapidly developing and that has many applications as well as ethical and practical implications. This article is a review that outlines the history of current technology, as well as the advantages and challenges in use for somatic cell and germline gene editing. Clustered regularly interspaced short palindromic repeat (CRISPR) was first introduced in 2002 and initially saw limited use among researchers. However, in 2012, it was discovered that a nuclease associated with CRISPR (Cas9) could be used with guide RNA to cleave double-stranded DNA in specific places, raising the potential for targeted gene editing. Immediately, the scientific community saw endless possibilities of this technology for clinical therapies and treatment of genetic disorders. Currently, more than 110 gene editing–based therapies are in clinical trials, with 2 different treatments already in phase 3 clinical trials. To date, all gene editing therapies that have progressed to clinical trials are somatic cell editing therapies. Germline gene editing, in which changes to genes can be passed to the next generation, is attractive for inherited diseases but has many ethical considerations and challenges that must be addressed before this technology is further developed. One concern is off-target editing events that could involve DNA sequences with high similarity to the target sequence. In addition to this, DNA breaks from Cas9 can result in chromosomal instability and possible abnormalities such as large deletions, translocations, and rearrangements. This could be problematic particularly in germline editing, where the effects are passed down to the next generation. Gene editing does, however, have great potential. Somatic cell gene editing has been successfully used to prevent and treat disease. One advantage of somatic cells is that they can be removed, edited, and returned to the patient to proliferate normally. For the therapies currently in stage 3 clinical trials, there have been few instances of failure to modify the alleles and no evidence of off-target editing using this method. Somatic cell gene editing has successfully been used in animal models and in limited human trials to treat diseases with easily accessible in vivo sites, such as the eye. Germline gene editing has advantages over somatic cell gene editing, as therapies can be developed to permanently correct heritable genetic diseases. It could also be adapted easily during an in vitro fertilization process for parents who are carriers of 1 or more diseases to eliminate the risk of their offspring manifesting the disease. However, germline editing creates changes to the DNA that is then passed to offspring, raising ethical and safety concerns. Many trials in animal models as well as early human clinical trials show promise for these treatments, but challenges remain including costs and logistical considerations such as time, space, and expertise. Another challenge is that because these therapies are targeted, each variation of each disease must be treated differently, with a therapy developed specifically for each disease and potentially even variations of the same disease. This again represents significant challenges related to cost, time, and expertise. Ethical challenges for germline gene editing include the use of human embryos, high rates of off-target editing, and possible severe chromosomal deletions depending on the target. Large chromosomal rearrangements, as required with attempting to eradicate certain diseases, have been recently shown to be more difficult and dangerous than smaller rearrangements. This presents a unique challenge that must be overcome before implementing this technique in any therapies. There are many possibilities and exciting opportunities with gene editing therapies, some of which are closer to fruition than others. We are potentially close to implementing some therapies, such as those for sickle cell disease, while farther from broad use for other therapies. The pace at which this technology has developed is astounding, which brings hope that this research will continue to progress quickly, and we may be closer than we think to effectively treating genetic disease with gene editing technology.

DNA- and selectable-marker-free genome-editing system using zygotes from recalcitrant maize inbred B73.

Genome-editing tools such as the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system have become essential tools for increasing the efficiency and accuracy of plant breeding. Using such genome-editing tools on maize, one of the most important cereal crops of the world, will greatly benefit the agriculture and the mankind. Conventional genome-editing methods typically used for maize involves insertion of a Cas9-guide RNA expression cassette and a selectable marker in the genome DNA; however, with such methods, it is essential to eliminate the inserted DNA cassettes to avoid legislative concerns on gene-modified organisms. Another major hurdle for establishing an efficient and broadly applicable DNA-free genome-editing system for maize is presented by recalcitrant genotypes/cultivars, since cell/tissue culture and its subsequent regeneration into plantlets are crucial for producing transgenic and/or genome-edited maize. In this study, to establish a DNA-free genome-editing system for recalcitrant maize genotypes/cultivars, Cas9-gRNA ribonucleoproteins (RNPs) were directly delivered into zygotes isolated from the pollinated flowers of the maize-B73 cultivar. The zygotes were successfully developed and regenerated into genome-edited plantlets by co-culture with phytosulfokine, a peptide phytohormone. The method developed herein made it possible to obtain DNA- and selectable-marker-free genome-edited recalcitrant maize genotypes/cultivars with high efficiency. This method can advance the molecular breeding of maize and other important cereals, regardless of their recalcitrant characteristics.

CRISPR- Cas9 Technology: Mechanism and Its Application in The Field of Entomology

The field of life science research has undergone a revolution thanks to the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated protein (CRISPR/Cas) system, which provides a multitude of opportunities for modifying, identifying, visualising, and annotating particular DNA or RNA sequences in diverse organisms. In this technique, foreign DNA pieces, known as spacers, are inserted into CRISPR cassettes. These spacers are then transcribed into CRISPR arrays and processed to produce guide RNA (gRNA). The Cas proteins that the CRISPR arrays encode serve as the enzymatic machinery required to obtain new spacers that specifically target invasive genetic elements. Several Cas proteins, such as Cas9, Cas12, Cas13, and Cas14, have been used to create novel tools for genome engineering due to their programmable sequence specificity. The ability to manipulate and edit nucleic acid sequences in living cells from a wide variety of organisms has been made possible by these Cas variants, which have greatly advanced genetic research and the CRISPR/Cas tool. The CRISPR Cas-9 technology has applications in many areas of entomology, including the genetics of honeybees and plants that produce insecticidal compounds. CRISPR/Cas9 technology has transformed entomology by providing precise tools for gene editing and genetic manipulation in insects. This has enabled advancements in fundamental research, disease vector control, and pest management, with the potential to reduce the environmental and economic impact of insect pests in agriculture and public health.

Region-Specific Phosphorylation Determines Neuroligin-3 Localization to Excitatory Versus Inhibitory Synapses

BackgroundNeuroligin-3 is a postsynaptic adhesion molecule involved in synapse development and function. It is implicated in rare, monogenic forms of autism, and its shedding is critical to the tumor microenvironment of gliomas. While other members of the neuroligin family exhibit synapse-type specificity in localization and function through distinct interactions with postsynaptic scaffold proteins, the specificity of neuroligin-3 synaptic localization remains largely unknown. MethodsWe investigated the synaptic localization of neuroligin-3 across regions in mouse and human brain samples after validating antibody specificity in knockout animals. We raised a phospho-specific neuroligin antibody and used phosphoproteomics, cell-based assays, and in utero CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9) knockout and gene replacement to identify mechanisms that regulate neuroligin-3 localization to distinct synapse types. ResultsNeuroligin-3 exhibits region-dependent synapse specificity, largely localizing to excitatory synapses in cortical regions and inhibitory synapses in subcortical regions of the brain in both mice and humans. We identified specific phosphorylation of cortical neuroligin-3 at a key binding site for recruitment to inhibitory synapses, while subcortical neuroligin-3 remained unphosphorylated. In vitro, phosphomimetic mutation of that site disrupted neuroligin-3 association with the inhibitory postsynaptic scaffolding protein gephyrin. In vivo, phosphomimetic mutants of neuroligin-3 localized to excitatory postsynapses, while phospho-null mutants localized to inhibitory postsynapses. ConclusionsThese data reveal an unexpected region-specific pattern of neuroligin-3 synapse specificity, as well as a phosphorylation-dependent mechanism that regulates its recruitment to either excitatory or inhibitory synapses. These findings add to our understanding of how neuroligin-3 is involved in conditions that may affect the balance of excitation and inhibition.

Effect of TRAF6-knockout on gene expression and lncRNA expression in Epithelioma papulosum cyprini (EPC) cells

ABSTRACT TRAF6 is a key immune gene that plays a significant role in toll-like receptor signal transduction and activates downstream immune genes involved in antiviral immunity in fish. To explore the role of TRAF6 in Epithelioma papulosum cyprini (EPC) cells, we knocked out the TRAF6 gene using the Clustered Regularly Interspaced Short Palindromic Repeats-Cas9 (CRISPR-Cas9) technique and then analyzed the transcriptomes of the knockout cells. In this study, we identified that 232 transcripts were differentially expressed in naive cells. Using the pipeline, we identified 381 novel lncRNAs in EPC cells, 23 of which were differentially expressed. Gene Ontology enrichment analysis demonstrated that differentially expressed genes (DEG) are implicated in various immune processes, such as neutrophil chemotaxis and mitogen-activated protein kinase binding. In addition, the KEGG pathway analysis revealed enrichment in immune-related pathways (Interleukin-17 signaling pathway, cytokine-cytokine receptor interaction, and TNF signaling pathway). Furthermore, the target genes of the differentially expressed lncRNAs were implicated in the negative regulation of interleukin-6 and tumor necrosis factor production. These results indicate that lncRNAs and protein-coding genes participate in the regulation of immune and metabolic processes in fish.

Applications of CRISPR Cas-9 in Ovarian Cancer Research.

Ovarian cancer is a highly prevalent malignancy among women and affects a significant population worldwide. Different forms of hormonal treatments or chemotherapies are used to treat ovarian cancer, but the possible side effects, including menopausal symptoms, can be severe, forcing some patients to prematurely stop the treatment. The emerging genome editing technology, known as clustered regularly interspaced short palindromic repeats (CRISPR)-caspase 9 (Cas9), has the potential to treat ovarian cancer via gene editing strategies. Studies have reported CRISPR knockouts of several oncogenes that are involved in the pathogenesis of ovarian cancer, such as BMI1, CXCR2, MTF1, miR-21, and BIRC5, and demonstrate the potential of the CRISPR-Cas9 genome editing technique to effectively treat ovarian cancer. However, there are limitations that restrict the biomedical applications of CRISPR-Cas9 and limit the implementation of Gene therapy for ovarian cancer. These include off-target DNA cleavage and the effects of CRISPR-Cas9 in non-target, normal cells. This article aims to review the current state of ovarian cancer research, highlight the significance of CRISPR-Cas9 in ovarian cancer treatment, and establish the groundwork for potential clinical research.

Improvement of resistance to rice blast and bacterial leaf streak by CRISPR/Cas9-mediated mutagenesis of Pi21 and OsSULTR3;6 in rice (Oryza sativa L.).

Rice (Oryza sativa L.) is a staple food in many countries around the world, particularly in China. The production of rice is seriously affected by the bacterial leaf streak and rice blast, which can reduce rice yield or even cause it to fail to be harvested. In this study, susceptible material 58B was edited by CRISPR/Cas9, targeting a target of the Pi21 gene and a target of the effector-binding element (EBE) of the OsSULTR3;6 gene, and the mutants 58b were obtained by Agrobacterium-mediated method. The editing efficiency of the two targets in the T0 generation was higher than 90.09%, the homozygous mutants were successfully selected in the T0 generation, and the homozygous mutation rate of each target was higher than 26.67%. The expression of the edited pi21 and EBE of Ossultr3;6 was significantly reduced, and the expression of defense responsive genes was significantly upregulated after infected with rice blast. The lesion areas of rice blast and bacterial leaf streak were significantly reduced in 58b, and the resistance of both was effectively improved. Furthermore, the gene editing events did not affect the agronomic traits of rice. In this study, the resistance of 58b to rice blast and bacterial leaf streak was improved simultaneously. This study provides a reference for using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) to accelerate the improvement of rice varieties and the development of new materials for rice breeding.

RF02 Using functional genomic techniques to help us understand the TYK2 pathway in psoriasis

Abstract Genetic screens have identified around 126 genetic signals associated with psoriasis through genome-wide association studies (GWAS) and rare variant analyses (Tsoi LC, Stuart PE, Tian C et al. Large scale meta-analysis characterizes genetic architecture for common psoriasis associated variants. Nat Commun 2017; 8:15382). The aim of this study was to use genome-editing techniques to investigate and validate the effect of a psoriasis-associated genetic signal on the expression of TYK2. GWAS have linked TYK2 to psoriasis susceptibility through the association of a protein-changing, exonic variant where TYK2 impairment confers protection against the development of psoriasis and other autoimmune diseases. The vast majority of disease risk-associated variants are found outside gene-coding regions. Functional experimental techniques may help characterize the effect of these variants on gene expression. The development of genome editing technology, clustered regularly interspaced short-palindromic repeat-Cas9 (CRISPR-Cas9), has made it possible to edit cell genomes. We used CRISPR-Cas9 to validate functionally the psoriasis-associated single nucleotide polymorphism (SNP) rs892085 on TYK2 expression in immortal T-cell lines. In-house data from our centre revealed the presence of long-range physical interactions between rs892085 and TYK2. We are the first to investigate functionally this signal and characterize this physical connection. We used CRISPR-Cas9 systems to both activate and inhibit the region around rs892085 and then measured the consequence of this through informative functional readouts using quantitative reverse transcription polymerase chain reaction. This revealed consistent alteration of TYK2 expression upon inhibition and activation of rs892085. This supports previous observations, where this regulatory region harbouring GWAS-implicated variation operates through direct long-distance contact. Previously, the closest or most biologically relevant gene was often assigned to a GWAS variant. We have shown that this is unreliable, with DNA regions looping over large distances to control genes. This work provides the first functional validation of the psoriasis-associated SNP rs892085. TYK2 is a recognized drug target with one therapy, deucravacitinib, approved by the US Food and Drug Administration. Drugs entering the clinical development stage that have used human genetic data to select drug targets may be more likely to succeed. This work demonstrates the potential complexity of how risk alleles may work to dysregulate TYK2 signalling in disease, with both gene function and expression increasing the risk of disease. Ultimately, genetic risk scores, incorporating functional evidence from studies like this, may be generated to predict how patients with different genetically perturbed biology pathways may respond to therapy.

Metabolic engineering of Bacillus subtilis toward the efficient and stable production of C30-carotenoids

Commercial carotenoid production is dominated by chemical synthesis and plant extraction, both of which are unsustainable and can be detrimental to the environment. A promising alternative for the mass production of carotenoids from both an ecological and commercial perspective is microbial synthesis. To date, C30 carotenoid production in Bacillus subtilis has been achieved using plasmid systems for the overexpression of biosynthetic enzymes. In the present study, we employed a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system to develop an efficient, safe, and stable C30 carotenoid-producing B. subtilis strain, devoid of plasmids and antibiotic selection markers. To this end, the expression levels of crtM (dehydrosqualene synthase) and crtN (dehydrosqualene desaturase) genes from Staphylococcus aureus were upregulated by the insertion of three gene copies into the chromosome of B. subtilis. Subsequently, the supply of the C30 carotenoid precursor farnesyl diphosphate (FPP), which is the substrate for CrtMN enzymes, was enhanced by expressing chromosomally integrated Bacillus megaterium-derived farnesyl diphosphate synthase (FPPS), a key enzyme in the FPP pathway, and abolishing the expression of farnesyl diphosphate phosphatase (YisP), an enzyme responsible for the undesired conversion of FPP to farnesol. The consecutive combination of these features resulted in a stepwise increased production of C30 carotenoids. For the first time, a B. subtilis strain that can endogenously produce C30 carotenoids has been constructed, which we anticipate will serve as a chassis for further metabolic engineering and fermentation optimization aimed at developing a commercial scale bioproduction process.

Strategies for Using CRISPR-Cas9 Orthologs to Perform Gene Editing Applications

CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats -Cas9) is primarily the adaptive immune system developed by bacteria and archaea to defend themselves from an invasion of the virus. CRISPR-Cas9 is considered currently the most potential gene editing technology in 21 century due to its highest operationality and modifiability among the gene-editing tech that humans have ever found. Orthologous proteins are the proteins of different species which originally develop from one ancestor and still hold the same function as the original protein. This research mainly concludes and organizes the strategies for two specific types of Cas9 orthologs in the application of gene editing.

Constitutive and conditional gene knockout mice for the study of intervertebral disc degeneration: Current status, decision considerations, and future possibilities.

There have been an increasing number of patients with degenerative disc diseases due to the aging population. In light of this, studies on the pathogenesis of intervertebral disc degeneration have become a hot topic, and gene knockout mice have become a valuable tool in this field of research. With the development of science and technology, constitutive gene knockout mice can be constructed using homologous recombination, zinc finger nuclease, transcription activator-like effector nuclease technology and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system, and conditional gene knockout mice can be constructed using the Cre/LoxP system. The gene-edited mice using these techniques have been widely used in the studies on disc degeneration. This paper reviews the development process and principles of these technologies, functions of the edited genes in disc degeneration, advantages, and disadvantages of different methods and possible targets of the specific Cre recombinase in intervertebral discs. Recommendations for the choice of suitable gene-edited model mice are presented. At the same time, possible technological improvements in the future are also discussed.

Single-cell resolution reveals RalA GTPase expanding hematopoietic stem cells and facilitating of BCR-ABL1-driven leukemogenesis in a CRISPR/Cas9 gene editing mouse model.

BCR-ABL oncogene-mediated Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) is suggested to originate from leukemic stem cells (LSCs); however, factors regulating self-renewal of LSC and normal hematopoietic stem cells (HSCs) are largely unclear. Here, we show that RalA, a small GTPase in the Ras downstream signaling pathway, has a critical effect on regulating the self-renewal of LSCs and HSCs. A RalA knock-in mouse model (RalARosa26-Tg/+) was initially constructed on the basis of the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) assay to analyze normal hematopoietic differentiation frequency using single-cell resolution and flow cytometry. RalA overexpression promoted cell cycle progression and increased the frequency of granulocyte-monocyte progenitors (GMPs), HSCs and multipotent progenitors (MPPs). The uniform manifold approximation and projection (UMAP) plot revealed heterogeneities in HSCs and progenitor cells (HSPCs) and identified the subclusters of HSCs and GMPs with a distinct molecular signature. RalA also promoted BCR-ABL-induced leukemogenesis and self-renewal of primary LSCs and shortened the survival of leukemic mice. RalA knockdown prolonged survival and promoted sensitivity to imatinib in a patient-derived tumor xenograft model. Immunoprecipitation plus single-cell RNA sequencing of the GMP population confirmed that RalA induced this effect by interacting with RAC1. RAC1 inhibition by azathioprine effectively reduced the self-renewal, colony formation ability of LSCs and prolonged the survival in BCR-ABL1-driven RalA overexpression CML mice. Collectively, RalA was detected to be a vital factor that regulates the abilities of HSCs and LSCs, thus facilitating BCR-ABL-triggered leukemia in mice. RalA inhibition serves as the therapeutic approach to eradicate LSCs in CML.

Patient-Induced Pluripotent Stem Cell–Derived Hepatostellate Organoids Establish a Basis for Liver Pathologies in Telomeropathies

Dyskeratosis congenita (DC) is a telomere biology disorder caused primarily by mutations in the DKC1 gene. Patients with DC and related telomeropathies resulting from premature telomere dysfunction experience multiorgan failure. In the liver, DC patients present with nodular hyperplasia, steatosis, inflammation, and cirrhosis. However, the mechanism responsible for telomere dysfunction-induced liver disease remains unclear. We used isogenic human induced pluripotent stem cells (iPSCs) harboring a causal DC mutation in DKC1 or a CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9)-corrected control allele to model DC liver pathologies. We differentiated these iPSCs into hepatocytes (HEPs) or hepatic stellate cells (HSCs) followed by generation of genotype-admixed hepatostellate organoids. Single-cell transcriptomics were applied to hepatostellate organoids to understand cell type-specific genotype-phenotype relationships. Directed differentiation of iPSCs into HEPs and stellate cells and subsequent hepatostellate organoid formation revealed a dominant phenotype in the parenchyma, withDC HEPs becoming hyperplastic and also eliciting a pathogenic hyperplastic, proinflammatory response in stellate cells independent of stellate cell genotype. Pathogenic phenotypes in DKC1-mutant HEPs and hepatostellate organoids could be rescued via suppression of serine/threonine kinase AKT (protein kinase B) activity, a central regulator of MYC-driven hyperplasia downstream of DKC1 mutation. Isogenic iPSC-derived admixed hepatostellate organoids offer insight into the liver pathologies in telomeropathies and provide a framework for evaluating emerging therapies.

Genome editing technologies, mechanismsand improved production of therapeutic phytochemicals: Opportunities and prospects.

Plants produce a large number of secondary metabolites, known as phytometabolites that may be employed as medicines, dyes, poisons, and insecticides in the field of medicine, agriculture, and industrial use, respectively. The rise of genome management approaches has promised a factual revolution in genetic engineering. Targeted genome editing in living entities permits the understanding of the biological systems very clearly, and also sanctions to address a wide-ranging objective in the direction of improving features of plant and their yields. The last few years have introduced a number of unique genome editing systems, including transcription activator-like effector nucleases, zinc finger nucleases, and miRNA-regulated clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). Genome editing systems have helped in the transformation of metabolic engineering, allowing researchers to modify biosynthetic pathways of different secondary metabolites. Given the growing relevance of editing genomes in plant research, the exciting novel methods are briefly reviewed in this chapter. Also, this chapter highlights recent discoveries on the CRISPR-based modification of natural products in different medicinal plants.

Interruption of aberrant chromatin looping is required for regenerating RB1 function and suppressing tumorigenesis

RB transcriptional corepressor 1 (RB1) is a critical regulatory gene in physiological and pathological processes. Genetic mutation is considered to be the main cause of RB1 inactivation. However, accumulating evidence has shown that not all RB1 dysfunction is triggered by gene mutations, and the additional mechanism underlying RB1 dysfunction remains unclear. Here, we firstly reveal that a CCCTC binding factor (CTCF) mediated intrachromosomal looping served as a regulatory inducer to inactivate RB1. Once the core genomic fragment was deleted by Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9), this intrachromosomal looping was disrupted. After the open of chromatin, Enhancer of Zeste Homolog 2 (EZH2) was released and decreased the level of Tri-Methyl-Histone H3 Lys27 (H3K27me3) at the RB1 promoter, which substantially restored the expression of RB protein (pRB) and inhibited tumorigenesis. In addition, targeted correction of abnormal RB1 looping using the small-molecule compound GSK503 efficiently restored RB1 transcription and suppressed tumorigenesis. Our study reveals an alternative transcriptional mechanism underlying RB1 dysfunction independent of gene mutation, and advancing the discovery of potential therapeutic chemicals based on aberrant chromatin looping.

Ethanol yield improvement in Saccharomyces cerevisiae GPD2 Delta FPS1 Delta ADH2 Delta DLD3 Delta mutant and molecular mechanism exploration based on the metabolic flux and transcriptomics approaches

BackgroundSaccharomyces cerevisiae generally consumes glucose to produce ethanol accompanied by the main by-products of glycerol, acetic acid, and lactic acid. The minimization of the formation of by-products in S. cerevisiae was an effective way to improve the economic viability of the bioethanol industry. In this study, S. cerevisiae GPD2, FPS1, ADH2, and DLD3 genes were knocked out by the Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR-Cas9) approach. The mechanism of gene deletion affecting ethanol metabolism was further elucidated based on metabolic flux and transcriptomics approaches.ResultsThe engineered S. cerevisiae with gene deletion of GPD2, FPS1, ADH2, and DLD3 was constructed by the CRISPR-Cas9 approach. The ethanol content of engineered S. cerevisiae GPD2 Delta FPS1 Delta ADH2 Delta DLD3 Delta increased by 18.58% with the decrease of glycerol, acetic acid, and lactic acid contents by 22.32, 8.87, and 16.82%, respectively. The metabolic flux analysis indicated that the carbon flux rethanol in engineered strain increased from 60.969 to 63.379. The sequencing-based RNA-Seq transcriptomics represented 472 differential expression genes (DEGs) were identified in engineered S. cerevisiae, in which 195 and 277 genes were significantly up-regulated and down-regulated, respectively. The enriched pathways of up-regulated genes were mainly involved in the energy metabolism of carbohydrates, while the down-regulated genes were mainly enriched in acid metabolic pathways.ConclusionsThe yield of ethanol in engineered S. cerevisiae increased with the decrease of the by-products including glycerol, acetic acid, and lactic acid. The deletion of genes GPD2, FPS1, ADH2, and DLD3 resulted in the redirection of carbon flux.

A zwitterionic polymer-inspired material mediated efficient CRISPR-Cas9 gene editing

The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR/Cas9) adaptive immune system is a cutting-edge genome-editing toolbox. However, its applications are still limited by its inefficient transduction. Herein, we present a novel gene vector, the zwitterionic polymer-inspired material with branched structure (ZEBRA) for efficient CRISPR/Cas9 delivery. Polo-like kinase 1 (PLK1) acts as a master regulator of mitosis and overexpresses in multiple tumor cells. The Cas9 and single guide sgRNA (sgRNA)-encoded plasmid was transduced to knockout Plk1 gene, which was expected to inhibit the expression of PLK1. Our studies demonstrated that ZEBRA enabled to transduce the CRISPR/Cas9 system with large size into the cells efficiently. The transduction with ZEBRA was cell line dependent, which showed ∼10-fold higher in CD44-positive cancer cell lines compared with CD44-negative ones. Furthermore, ZEBRA induced high-level expression of Cas9 proteins by the delivery of CRISPR/Cas9 and efficient gene editing of Plk1 gene, and inhibited the tumor cell growth significantly. This zwitterionic polymer-inspired material is an effective and targeted gene delivery vector and further studies are required to explore its potential in gene delivery applications.

CRISPR/Cas9 Technique for Temperature, Drought, and Salinity Stress Responses

Global warming and climate change have severely affected plant growth and food production. Therefore, minimizing these effects is required for sustainable crop yields. Understanding the molecular mechanisms in response to abiotic stresses and improving agricultural traits to make crops tolerant to abiotic stresses have been going on unceasingly. To generate desirable varieties of crops, traditional and molecular breeding techniques have been tried, but both approaches are time-consuming. Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) and transcription activator-like effector nucleases (TALENs) are genome-editing technologies that have recently attracted the attention of plant breeders for genetic modification. These technologies are powerful tools in the basic and applied sciences for understanding gene function, as well as in the field of crop breeding. In this review, we focus on the application of genome-editing systems in plants to understand gene function in response to abiotic stresses and to improve tolerance to abiotic stresses, such as temperature, drought, and salinity stresses.