In today’s rapidly evolving digital landscape, the significance of password guessing techniques in both offensive and defensive strategies is paramount. Passwords serve as a crucial line of defense for both individuals and corporations, safeguarding their sensitive systems and data. Therefore, assessing the effectiveness of these access credentials is a critical task. However, existing research in this field often encounters limitations, such as a lack of sufficient training data and extended model training times. Current methods often struggle with limited training data and lengthy training times. This paper introduces BBPE-AE, a novel Auto Encoder Network (AE) designed for password guessing. BBPE-AE utilizes Byte-level Byte Pair Encoding (BBPE) to extract frequent tokens from password datasets without length restrictions, employing a dynamic window technique to capture complex patterns. Experimental results on Hotmail and Myspace datasets demonstrate exceptional performance, achieving high similarity rates (BLEU-Unigram: 0.90, BLEU-Bigram: 0.82 for Hotmail; BLEU-Unigram: 0.90, BLEU-Bigram: 0.81 for Myspace). BBPE-AE generates realistic passwords that meet HIBP (Have I Been Pwned) standards, minimizing duplication. These findings highlight the effectiveness of BBPE-AE in enhancing security by generating realistic passwords, ultimately safeguarding sensitive systems and data.

The utilization of electroporation for delivering the CRISPR/Cas9 system components has enabled efficient gene editing in mammalian zygotes, facilitating the development of genome-edited animals. In our study，This research focused on targeting the ACTG1 and MSTN genes in sheep, revealing a threshold phenomenon in electroporation with a voltage tolerance in sheep in vitro fertilization (IVF) zygotes. Various poring voltages near 40V and pulse durations were examined for electroporating sheep zygotes. The study concluded that stronger electric fields required shorter pulse durations to achieve the optimal conditions for high gene mutation rates and reasonable blastocyst development. The investigation also assessed the quality of Cas9/sgRNA ribonucleoprotein complexes (Cas9 RNPs) and their influence on genome editing efficiency in sheep early embryos. It was highlighted that pre-complexation of Cas9 proteins with single-guide RNA (sgRNA) before electroporation was essential for achieving a high mutation rate. The use of suitable electroporation parameters for sheep IVF zygotes led to significantly high mutation rates and heterozygote ratios. By delivering Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) to zygotes through electroporation, targeting the MSTN (Myostatin) gene, a knock-in efficiency of 26% was achieved. The successful generation of MSTN-modified lambs was demonstrated by delivering Cas9 RNPs into IVF zygotes via electroporation.

Background/Objectives: Direct oral anticoagulants (DOACs) are first-line medications for stroke prevention in non-valvular atrial fibrillation (AF). However, variability in drug response poses risks of hemorrhagic or thromboembolic events. The role of genetics in determining DOAC safety is gaining attention, but evidence linking specific genetic polymorphisms to bleeding risk is lacking. Therefore, the aim of our study was to conduct an Exome-Wide Association Study with 198 non-valvular AF patients treated with rivaroxaban or apixaban, including 97 with bleeding complications and 99 without. Methods: DOAC plasma concentrations, 6-β-hydroxycortisol and cortisol levels in urine were measured for CYP3A4 phenotyping. Sequencing was performed on the DNBSEQ G-400 platform. Single nucleotide variants (SNV) associations with bleeding risk were assessed using logistic regression with additive, dominant, and recessive genetic models. Results: No SNVs reached Bonferroni-corrected significance in any genetic model. Polygenic risk scores (PRS) showed weak predictive ability for bleeding with apixaban. For rivaroxaban, regression indicated that ln Css min/D + 1 index increased with PRS, age, and 6-β-hydroxycortisol/cortisol ratio, but decreased with higher 6-β-hydroxycortisol and coronary heart disease presence. Conclusions: The findings revealed that the residual equilibrium concentration of anticoagulants, including dose-adjusted, cannot alone predict bleeding risk in non-valvular AF patients. No new genetic variants associated with bleeding risk were identified.

Scientists can use the Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 system, which is a powerful and flexible tool for changing the genome, to change only certain parts of the genome. This has helped researchers figure out how target genes work in biology and disease. CRISPR/Cas9 is made up of a sequence-specific CRISPR RNA (crRNA) set and a generic Cas9 nuclease. The crRNA set tells Cas9 where to cut DNA and cause double-strand breaks. In the next step of cellular DNA repair, insertions, deletions, and swaps are made at specific spots. For CRISPR/Cas9-mediated DNA cleavage to be specific, the target sequences must match the crRNA, and a protospacer nearby motif must be placed after the target sequences. In this article, we talk about how CRISPR/Cas9 works at the molecular level, what it can be used for, what it can't do, and what its future clinical treatment promise might be.

This study aimed to identify miRNA editing sites during adipose tissue development in Ningxiang pigs and analyze their characteristics and functions. Based on the small RNA-seq data from subcutaneous adipose tissue samples of Ningxiang pigs at four stages (30, 90, 150 and 210 days), we constructed a developmental atlas of the miRNA editome in adipose tissue of Ningxiang pigs. The characteristics and functions of this editome were further explored through bioinformatics analysis. The findings revealed that a total of 505 miRNA editing sites were identified with C-to-U editing types being the most prevalent, followed by U-to-C, A-to-G and G-to-U. Importantly, these four types of miRNA editing exhibited a base preference. The number of editing sites showed obvious differences among age groups, with the highest occurrence of miRNA editing events observed at 90 days of age and the lowest at 150 days of age. In the miRNA seed region, a total of 9 miRNA editing sites were identified with significant differences in editing levels (P < 0.05), and located in ssc-miR-23a, ssc-miR-27a, ssc-miR-30b-5p, ssc-miR-15a, ssc-miR-497, ssc-miR-15b, and ssc-miR-425-5p, respectively. Target gene prediction and enrichment analysis revealed that the editing of miR-497 may potentially regulate fat deposition by inhibiting adipose synthesis via influencing target binding. These results provide new insights into the regulatory mechanism of pig fat deposition.

Abiotic stress, especially drought stress poses a significant threat to the terrestrial plant growth, development, and productivity. Although mulberry have great genetic diversity and extensive stress tolerant traits in agroforestry systems, only a few reports offer preliminary insight into biochemical responses of mulberry leaves under drought conditions. In this study, we performed a comparative metabolomic and transcriptomic analysis on the “drooping mulberry” (Morus alba var. Pendula Dippel) under PEG-6000-simulated drought stress. Our research revealed that drought stress significantly enhanced flavonoid accumulation and up-regulated the expression of phenylpropanoid biosynthetic genes. Furthermore, the activities of superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) content were elevated. In vitro enzyme assays and fermentation tests indicated the involvement of flavonol synthase/flavanone 3-hydroxylase (XM_010098126.2) and anthocyanidin 3-O-glucosyltransferase 5 (XM_010101521.2) in the biosynthesis of flavonol aglycones and glycosides, respectively. The recombinant MaF3GT5 protein was found to recognize kaempferol, quercetin, and UDP-glucose as substrates, but not 3-/7-O-glucosylated flavonols and UDP-rhamnose. MaF3GT5 is capable of forming 3-O- and 7-O-monoglucoside, but not di-O-glucosides, form kaempferol. This implies its role as a flavonol 3, 7-O-glucosyltransferase. The findings from this study provided insights into the biosynthesis of flavonoids and could have substantial implications for the future diversified utilization of mulberry.

The flowering period of rice significantly impacts variety adaptability and yield formation. Properly shortening the reproductive period of rice varieties can expand their ecological range without significant yield reduction. Targeted genome editing, like CRISPR/Cas9, is an ideal tool to fine-tune rice growth stages and boost yield synergistically. In this study, we developed a CRISPR/Cas9-mediated multiplex genome editing vector containing five genes related to three traits: Hd2, Ghd7, and DTH8 (flowering stage genes), along with the recessive rice blast resistance gene Pi21 and the aromatic gene BADH2. This vector was introduced into the high-quality Geng rice variety in Zhejiang province, Jiahe212 (JH212), resulting in 34 T0 plants with various effective mutations. Among the 17 mutant T1 lines, several displayed diverse flowering dates, but most exhibited undesirable agronomic traits. Notably, three homozygous mutant lines (JH-C15, JH-C18, and JH-C31) showed slightly earlier flowering dates without significant differences in yield-related traits compared to JH212. Through special Hyg and Cas marker selection of T2 plants, we identified 7, 6, and 2 fragrant glutinous plants devoid of transgenic components, respectively. These single plants will serve as sib lines of JH212 and potential resources for breeding applications, including maintenance lines for Xian-Geng interspecific three-line hybrid rice. In summary, our research offers practical insights and potential resources for targeted improvement of Geng rice varieties, and would also promote the Geng rice varieties expending from the southward to the northward.

Inflammasomes are multimolecular complexes with potent inflammatory activity. As such, their activity is tightly regulated at the transcriptional and post-transcriptional levels. In this review, we present the transcriptional regulation of inflammasome genes from sensors (e.g NLRP3) to substrates (e.g. IL-1β). Lineage-determining transcription factors shape inflammasome responses in different cell types with profound consequences on the responsiveness to inflammasome-activating stimuli. Pro-inflammatory signals (sterile or microbial) have a key transcriptional impact on inflammasome genes, which is largely mediated by NF-κB and, that translates into higher antimicrobial immune responses. Furthermore, diverse intrinsic (e.g. circadian clock, metabolites) or extrinsic (e.g. xenobiotics) signals are integrated by signal-dependent transcription factors and chromatin structure changes to modulate transcriptionally inflammasome responses. Finally, anti-inflammatory signals (e.g. IL-10) counterbalance inflammasome genes induction to limit deleterious inflammation. Transcriptional regulations thus appear as the first line of inflammasome regulation to raise the defense level in front of stress and infections but also to limit excessive or chronic inflammation.

Cachexia is a complex metabolic syndrome characterized by loss of skeletal muscle, leading to a significant weight loss that impacts patient morbidity and mortality. Given the complexity of gene regulatory networks that control gene expression, our objective was to perform an integrative mRNA and miRNA profiling to identify genetic programs that capture essential mechanistic details that promote muscle atrophy in cancer cachexia. Here, we used RNA sequencing to analyze miRNAs and mRNAs expression profiles in tibialis anterior (TA) muscles of the Lewis lung carcinoma model of cancer cachexia. In addition, we compared these findings with RNA-Seq data from C2C12 myotubes treated in vitro with the cachectic factors tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). Extracellular matrix (ECM) alterations were validated by picrosirius staining, western blot, and fractal dimension analyses. We found 1,008 mRNAs and 18 miRNAs differentially expressed in cachectic mice. This set of genes was associated with the ECM, proteolysis, and inflammatory response. Enrichment analysis of transcriptional factor binding sites revealed activation of the atrophy-related transcriptional factors: NF-κB, Stat3, AP-1, and FoxO. Furthermore, the integration of mRNA and miRNA expression profiles identified post-transcriptional regulation by miRNAs of genes involved in ECM organization, cell migration, transcription factors binding, ion transport, and FoxO signaling pathway. C2C12 myotubes treated with TNF-α and IFN-γ similarly down-regulate subsets of ECM genes, including collagens. Our integrative analysis of miRNA-mRNA co-profiles comprehensive characterized regulatory relationships of molecular pathways and revealed miRNAs targeting ECM-associated genes in cancer cachexia. We also confirmed in C2C12 myotubes that changes in ECM-associated genes are dependent on inflammatory signaling of the cytokines TNF-α and IFN-γ.

Rice prolamins are categorized into three groups by molecular size (10, 13, or 16 kDa), while the 13 kDa prolamins are assigned to four subgroups (Pro13a-I, Pro13a-II, Pro13b-I, and Pro13b-II) based on cysteine residue content. Here, we generated four knockout strains of rice using CRISPR-Cas9, each of which demonstrated selectively reduced expression of specific subgroups of the 13 kDa prolamins. These four mutant rice lines also demonstrated compensatory expression of glutelins and non-targeted prolamins, which resulted in low grain weight, altered starch content and atypically-shaped starch granules and protein bodies. Transcriptome analysis identified 746 transcripts that were differentially expressed in the mutant rice lines during development. These transcripts played roles primarily in RNA processing, protein synthesis, stress response, and transport. After selective suppression/knockout of genes in the Pro13a-I subgroup, compensatory upregulation of genes in the Pro13a-II and Pro13b-I/II subgroups was observed. Compensatory expression of 9 ER stress and 17 transcription factor genes was also observed in mutant rice in which expression of 13 kDa prolamin genes was suppressed. Our results provide valuable insight into the regulatory mechanisms underlying rice seed development and the specific roles played by 13 kDa rice prolamins and their target genes.

Gene or genome editing (GE) revises, removes, or replaces a mutated gene at the DNA level; it is a tool. Gene therapy (GT) offsets mutations by introducing a "normal" version of the gene into the body while the diseased gene remains in the genome; it is a medicine. So far, no GE product has been approved, as opposed to 22 GT products that cost up to millions of dollars per dose. The FDA has recently added a guideline specific to gene editing that should be understood to enable faster development of GE products; at the same time, the FDA also needs to bring more clarification and make several amendments to this guideline to make it more rational.

Video editing is a high-required job, for it requires skilled artists or workers equipped with plentiful physical strength and multidisciplinary knowledge, such as cinematography, aesthetics. Thus gradually, more and more researches focus on proposing semi-automatical and even fully automatical solutions to reduce workloads. Since those conventional methods are usually designed to follow some simple guidelines, they lack flexibility and capability to learn complex ones. Fortunately, the advances of computer vision and machine learning make up the shortages of traditional approaches and make AI editing feasible. There is no survey to conclude those emerging researches yet. This paper summaries the development history of automatic video editing, and especially the applications of AI in partial and full workflows. We emphasizes video editing and discuss related works from multiple aspects: modality, type of input videos, methology, optimization, dataset, and evaluation metric. Besides, we also summarize the progresses in image editing domain, i.e., style transferring, retargeting, and colorization, and seek for the possibility to transfer those techniques to video domain. Finally, we give a brief conclusion about this survey and explore some open problems.

Cardiomyopathies are diseases of heart muscle, a significant percentage of which are genetic in origin. Cardiomyopathies can be classified as dilated, hypertrophic, restrictive, arrhythmogenic right ventricular or left ventricular non-compaction, although mixed morphologies are possible. A subset of neuromuscular disorders, notably Duchenne and Becker muscular dystrophies, are also characterized by cardiomyopathy aside from skeletal myopathy. The global burden of cardiomyopathies is certainly high, necessitating further research and novel therapies. Genome editing tools, which include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR) systems have emerged as increasingly important technologies in studying this group of cardiovascular disorders. In this review, we discuss the applications of genome editing in the understanding and treatment of cardiomyopathy. We also describe recent advances in genome editing that may help improve these applications, and some future prospects for genome editing in cardiomyopathy treatment.

Hypoxia, or reduced oxygen availability, has been studied extensively for its ability to activate specific genes. Hypoxia induced gene expression is mediated by the HIF transcription factors, although not exclusively so. Despite the great knowledge on the mechanisms by which hypoxia activates genes, much less is known about how hypoxia promotes gene repression. In this review, we discuss the potential mechanisms underlying hypoxia-induced transcriptional repression responses. We highlight HIF-dependent and independent mechanisms, but also the potential roles of dioxygenases with functions at the nucleosome and DNA level. Finally, we discuss recent evidence regarding the involvement of transcriptional repressor complexes in hypoxia.

Myocardial hypertrophy is the most common condition that accompanies heart changings in children. Transcriptional pathways regulating gene expression playing a critical role both in cardiac embryogenesis and in the pathogenesis of congenital hypertrophic cardiomyopathy, neonatal posthypoxic myocardial hypertrophy and congenital heart diseases. This paper describes the state of cardiac gene expression and potential pharmacological modulators at different transcriptional levels. An experimental model of perinatal cardiac hypoxia showed downregulated expression of genes responsible for cardiac muscle integrity and overexpressed genes associated with energy metabolism and apoptosis, which may provide a basis for a therapeutic approach. Current evidence suggests that RNA drugs, theaflavin, neuraminidase, proton pump and histone deacetylase inhibitors are promising pharmacological agents in progressive cardiac hypertrophy. The different points of application of the above drugs makes combined use possible, potentiating the effects of inhibition in specific signaling pathways. The special role of N-acetyl cysteine in both the inhibition of several signaling pathways and the reduction of oxidative stress was emphasized.

Estrogen receptor-positive (ER+) breast cancer is common among postmenopausal women and is frequently treated with Letrozole. Letrozole inhibits aromatase from synthesizing estrogen from androgens. Decreased estrogen slows the growth of tumors and can be an effective treatment. Letrozole resistance is increasingly being observed, which poses a unique problem for patients. We reanalyzed transcriptomic data comparing individuals who responded to Letrozole therapy (responders) to those who were resistant to treatment (non-responders). We identified SOX11 and S100A9 as two genes that significantly differed between these patients; with “PLK1 signaling events” being the most significant signaling pathway. We also identified PRDX4 and E2F8 as the top transcriptional biomarkers for ER+ treatment resistance. Many of the other significant gene products play a known role in ER+ breast cancer or other types of cancer, which partially validate our results. Several of the gene products we identified do not have a characterized role in ER+ breast cancer. Our analysis identified a set of genes that warrant further research to elucidate the molecular mechanism of Letrozole resistance in this patient population, as well as novel high-performing biomarkers. We anticipate that these findings could contribute to improved therapeutic outcomes in human patients.

Genetic switches can be utilized for many purposes in synthetic biology including the assembly of complex genetic circuits to achieve sophisticated cellular systems and the construction of biosensors for real-time monitoring of intracellular metabolite concentrations. Although genetic switches have mainly been developed in prokaryotes to date, eukaryotic genetic switches are increasingly being reported as both rational and irrational engineering technologies mature. In this review, we describe genetic switches in yeast based on synthetic transcription factors and/or synthetic promoters. We also discuss directed evolution technologies for the rapid and robust construction of yeast genetic switches.

Selenium is a trace metal essential to human health, and its deficiency has been related to, for instance, cardiovascular and myodegenerative diseases, infertility and osteochondropathy Kashin-Beck disease. It is incorporated as selenocysteine to selenoproteins, which protect against reactive oxygen and nitrogen species. They also participate in the activation of thyroid hormone, and play a role in immune system functioning. The synthesis and incorporation of selenocysteine occurs via a special mechanism, which differs from the one used for standard amino acids. The codon for selenocysteine is the regular in-frame stop codon, which can be passed by specific complex machinery participating in translation elongation and termination. This includes the presence of selenocysteine insertion sequence (SECIS) in the 3’-untranslated part of the selenoprotein mRNAs. Selenium deficiency is known to control both selenoprotein and non-selenoprotein transcriptomes. Nonsense-mediated decay is involved in the regulation of selenoprotein mRNA levels, both other mechanisms are also possible.

Adeno-associated viral (AAV) vectors can be used for gene delivery. AAV.CAP-Mac was recently developed; it can cross the blood-brain barrier and transduce cells throughout the CNS. However, some brain regions were not transduced in adult rhesus monkeys. Additionally, AAV vectors can only package 5 kb of DNA. Also, AAV vectors may be genotoxic and cytotoxic, especially at high doses. Finally, AAV vectors are very expensive to produce at sufficiently high titers for treatment. Off-the-shelf cell-based delivery systems wherein the cells can infiltrate the target tissue and be hyper-motile would be ideal. Also, mRNA-based gene editing would be a transient treatment, and thus be much safer.

RNA editing involves the insertion, deletion or substitution of single nucleotides within a RNA molecule, without altering the DNA sequence. Adenosine to inosine (A-to-I) editing consists of an RNA modification where single adenosines along the RNA sequence are converted into inosines. Such a biochemical transformation is catalyzed by enzymes belonging to the family of adenosine deaminases acting on RNA (ADARs) and occurs either co- or post-transcriptionally. Initially, the A-to-I RNA editing phenomenon was discovered and studied in messenger RNAs (mRNAs), where it can influence RNA splicing and cause the recoding of codon sequences. The employment of more powerful, high-throughput detection methods has recently revealed that A-to-I editing widely occurs in non-coding RNAs, including microRNAs (miRNAs). MiRNAs are a class of small regulatory non-coding RNAs (ncRNAs) acting as translation inhibitors, known to exert relevant roles in controlling cell cycle, proliferation, and cancer development. Indeed, a growing number of recent researches have evidenced the importance of miRNA editing in cancer biology by exploiting various detection and validation methods. Herein, we briefly overview early and currently available A-to-I miRNA editing detection and validation methods and discuss the significance of A-to-I miRNA editing in human cancer.

Clustered regularly interspaced short palindromic repeats (CRISPR) based gene-editing has begun to transform the treatment landscape of genetic diseases. The history of the discovery of CRISPR/CRISPR-associated (Cas) proteins/single guide RNA (sgRNA)-based gene-editing since the first report of repetitive sequences of unknown significance in 1987 is fascinating, instructive, and inspiring for future advances. The recent approval of CRISPR-Cas9-based gene therapy to treat patients with severe sickle cell anemia and transfusion-dependent beta thalassemia has renewed hope for treating other hematologic diseases, including patients with germline predisposition to hematologic malignancies, who would benefit greatly from the development of CRISPR-based gene therapies. The purpose of this manuscript is three-fold: first, a chronological description of the history of CRISPR-Cas9-sgRNA-based gene editing; second, a brief description of the current state of clinical research in hematologic diseases, including selected applications in treating hematologic diseases with CRISPR-based gene therapy; and third, the current progress in gene therapies in inherited hematologic diseases and bone marrow failure syndromes, to hopefully stimulate efforts towards developing these therapies for patients with inherited bone marrow failure syndromes and other inherited conditions with germline predisposition to hematologic malignancies.

Animals are distinguished by their complex developmental programs, in which transcription factors play essential roles in coordinating the regulation of the genome. Thus, it has been hypothesized that animal origins involved the evolution of increasingly complex transcriptional regulatory mechanisms that permit greater spatiotemporal control over gene expression. Here, we revisit this hypothesis in light of new genomic and functional data from diverse phylogenetically-relevant taxa, including early branching animals (sponges, ctenophores) and close relatives of animals (choanoflagellates, filastereans, ichthyosporeans). We argue that many of the mechanisms posited to explain animal transcriptional complexity, such as large increases in transcription factor numbers, new transcription factor families, and distal enhancers, did not factor significantly into animal origins. Instead, we propose that the re-purposing of pre-existing transcriptional regulatory modules through cis-regulatory evolution and gene duplication events, combined with new protein-protein interactions among TFs, may have been fundamental to the transcriptional regulatory architecture of the first animals.

Clonal evolution and cellular plasticity are the genetic and non-genetic driving forces of tumor heterogeneity that in turn determines the tumor cell response towards therapeutic drugs. Several lines of evidence suggest that therapeutic interventions foster the selection of drug resistant neural crest stem-like cells (NCSCs) that establish minimal residual disease (MRD) in melanoma. Here we established a dual reporter system enabling the tracking of NGFR expression and mRNA stability, providing insights into the maintenance of NCSC-states. We observed that the transcriptional reporter that contained a 1kb fragment of the human NGFR promoter was activated only in a minor subset (0.72±0.49%, range 0.3-1.5) and ~2-4% of A375 melanoma cells revealed stable NGFR mRNA. The combination of both reporters provided insights into phenotype switching and revealed that both cellular subsets gave rise to cellular heterogeneity. Moreover, whole transcriptome profiling and gene set enrichment analysis (GSEA) of the minor cellular subset revealed hypoxia-associated programs that might serve as potential drivers of an in vitro switching of NGFR-associated phenotypes and relapse of post-BRAF inhibitor treated tumors. Concordantly, we observed that the minor cellular subset increased in response to dabrafenib over time. In summary, our reporter-based approach provided insights into plasticity and identified a cellular subset that might be responsible for the establishment of MRD in melanoma.

The development of CRISPR associated proteins, such as Cas9, has led to increased accessibility and ease of use in genome editing. However, additional tools are needed to quantify and identify successful genome editing events in living animals. We developed a method to rapidly and quantitatively monitor gene editing activity non-invasively in living animals that also facilitates confocal microscopy and nucleotide level analyses at the end of study. Here we report a new CRISPR “footprinting” approach to activate luciferase and fluorescent proteins in mice as a function of gene editing. This system is based on experience with our prior Cre-detector system and is designed for Cas editors able to target LoxP including gRNAs including SaCas9 and ErCas12a [1, 2]. These CRISPRs cut specifically within LoxP, an approach that is a departure from previous gene editing in vivo activity detection techniques that targeted adjacent stop sequences. In this sensor paradigm, CRISPR activity was monitored non-invasively in living Cre reporter mice (FVB.129S6(B6)-Gt(ROSA)26Sortm1(Luc)Kael/J and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J, which will be referred to as LSL and mT/mG throughout the paper) after intramuscular or intravenous hydrodynamic plasmid injections, demonstrating utility in two diverse organ systems. The same genome-editing event was examined at the cellular level in specific tissues by confocal microscopy to determine the identity and frequency of successfully genome-edited cells. Further, SaCas9 induced targeted editing at efficiencies that were comparable to Cre recombinase demonstrating high effective delivery and activity in a whole animal. This work establishes genome editing tools and models to track CRISPR editing in vivo non-invasively and to fingerprint the identity of targeted cells. This approach also enables similar utility for any of the thousands of previously generated LoxP animal models.

Conventional plant breeding has contributed enormously towards feeding the world and has played crucial roles in the development of modern society. The conventional method creates variation by transferring genes between or within the species. In general, these methods are more expensive and takes more time, to overcome these limitations, new technology is required. Genome editing is a powerful tool for biotechnology applications, with the capacity to alter the function of any gene. With the availability of gene information for the majority of the traits, genome editing emerged as a potential to create a new variation with the introduction of any transgene. The important genome editing tools used nowadays are ZFNs, TALEN, Pentatricopeptide repeats protein, adenine base editor, RNA interference, and CRISPR/Cas9. These tools have opened a new era for crop improvement. Due to the complex genetic architecture of most traits, it is challenging to edit genes controlling them. To overcome these challenges, genome editing provides a broader perspective. Among the above-mentioned tools, CRISPR/Cas9 is the most powerful tool for gene editing. These technologies are being used to create abiotic and biotic resistance crop varieties.

With rapid advances in gene editing and gene therapy technologies, the development of genetic, cell, or protein-based cures to disease are no longer the realm of science fiction but that of today’s practice. The impact of these technologies are rapidly bringing them to the veterinary market as both enhanced therapeutics and towards modeling their outcomes for translational application. Simply put, gene editing enables scientists to modify an organism’s DNA a priori through the use of site-specific DNA targeting tools like clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). Gene therapy is a broader definition that encompasses the addition of exogenous genetic materials into specific cells to correct a genetic defect. More precisely, the U.S Food and Drug Administration (FDA) defines gene therapy as “a technique that modifies a person’s genes to treat or cure disease” by either (i) replacing a disease-causing gene with a healthy copy of the gene; (ii) inactivating a disease-causing gene that was not functioning properly; or (iii) introducing a new or modified gene into the body to help treat a disease. In some instances, this can be accomplished through direct transfer of DNA or RNA into target cells of interest or more broadly through gene editing. While gene therapy is possible through the simple addition of genetic information into cells of interest, gene editing allows the genome to be reprogrammed intentionally through the deletion of diseased alleles, reconstitution of wild type sequence, or targeted integration of exogenous DNA to impart new function. Cells can be removed from the body, altered, and reinfused, or edited in vivo. Indeed, manufacturing and production efficiencies in gene editing and gene therapy in the 21^st century has brought the therapeutic potential of in vitro and in vivo reprogrammed cells, to the front lines of therapeutic intervention (Brooks et al., 2016). For example, CAR-T cell therapy is revolutionizing hematologic cancer care in humans and is being translated to canines by us and others, and gene therapy trials are ongoing for mitral valve disease in dogs.

Climate change poses a serious threat to global agricultural activity and food production. To address this issue, plant genome editing technologies have been developed to provide an alternative solution for crop improvement. Unlike conventional breeding techniques (e.g., selective breeding and mutation breeding), modern genome editing tools offer more targeted and specific alterations of the plant genome to produce crops with desired traits, such as higher yield and/or stronger resilience to the changing environment. In this review, we discuss the current development and future applications of genome editing technologies in mitigating the impacts of biotic and abiotic stresses on agriculture. We focus specifically on the CRISPR/Cas system, which has been the center of attention in the last few years as a revolutionary genome-editing tool in various species. We also conducted a bibliographic analysis on CRISPR-related papers published from 2012 to 2021 (10 years) to identify trends and possible gaps in the CRISPR/Cas-related plant research. In addition, this review article outlines the current shortcomings and challenges of employing genome editing technologies in agriculture with notes on future prospective. We believe combining conventional and more innovative technologies in agriculture would be the key to optimizing crop improvement beyond the limitations of traditional agricultural practices.

In "genome editing," a type of genetic engineering, DNA is added, taken away, or changed in an organism's genome. Biomedicine, biotechnology, and synthetic biology, just to name a few, have all gained a lot from how often this method is used. Before the editing process can start, a Double-Strand Breaks (DSB) must be made in the DNA at a specific gene. Researchers have made nucleases, which are sometimes called "molecular scissors," to fix this DSB. Transcription activator-like effector nucleases (TALENs), Zinc-finger nucleases (ZFNs), and homing endonucleases are all examples of protein targets that have been studied and changed. The last part gave an overview of these study projects and explained how to make Cas12a for specific uses. The applications of Cas12a have been extensively studied in recent years, but the protein still holds a lot of promise as a treatment and screening tool. In order to give a quick review of CRISPR-Cas12a and its uses, we will briefly talk about the structure and function of the different parts of the reaction pathway that lead up to the catalysis of the target DNA. Cas12a uses a multistep process to make sure that it is selecting the right DNA. This is a good trait for a device that changes the DNA because it makes it less likely that something bad will happen. Even though data shows that a new CRISPR RNAs (crRNA) molecule can stop Cas12a from randomly destroying ssDNA, it may still hurt the host cell while trying to change the genome. The action of Cas12a catalysis, which is a powerful tool for changing the genome, needs to be changed so that it can be more easily controlled and managed. Using what we know about how Cas12a works, we have made mutants that are less active on ssDNA and more active on dsDNA. So far, only Cas9 and Cas12a, which are both part of the CRISPR family, have been used to change the genome. Because of how similar and different these two endonucleases are, CRISPR can now be used for multiple scientific purposes. Cas12a is better than Cas9 because it makes double-strand breaks (DSBs) that favor Homology-Directed Repair (HDR) over Non-homologous end joining (NHEJ) and can change more than one copy of the genome at the same time. Both Cas9 and Cas12a are being changed to make them better at detecting Protospacer Adjacent Motif (PAM) than they are in their natural state. This will make it possible to start focusing on more genes.

Poplar (Populus) is a genus of woody plants of value for the pulp industry, green energy, urban landscaping, soil phytoremediation and as an ornamental plant. Due to the increasing economic importance of poplar, there is a need to ensure its stable growth by increasing its resistance to pathogens, as one of the main problems in poplar cultivation is its susceptibility to numerous diseases. Currently, breeding poplars with reduced susceptibility to infections is receiving increasing attention. Genetic engineering can produce organisms with improved traits faster than traditional methods, and with the development of CRISPR/Cas-based genome editing systems, scientists have a new highly efficient tool at their disposal to create valuable genotypes. In this review, we summarize recent research data on poplar diseases, the biology of their pathogens, and how these plants resist pathogens. In the final section, we discuss genetic engineering, genome editing, and microbiome strategies that we believe are suitable for obtaining new improved poplar lines.

We are in a new chapter of crop and livestock improvement with the emergence of genome editing. This latest generation of molecular tools can be used to make targeted changes in a genome including insertions, deletions, and mutations. With new advances comes new risks for unintended changes and impacts, thus the need for appropriate risk assessment for product development and to inform regulatory measures. Though CRISPR/Cas has arisen as the predominant technology, there are multiple types of genome editing tools each with pros and cons depending on the organism and desired outcome. Furthermore, each editing tool differs in specificity as they may edit non-intended sites, referred to as off-target edits. The consensus of the agricultural editing community is to avoid off-target editing through design and detection, instead of determining whether off-target editing in each case is detrimental. The design of a targeting component, the tool chosen, and the identification of the edit(s) made are the critical factors in avoiding off-target edits and confirming intended edits in final products that are released commercially. The limited amount of head-to-head comparisons of genome editing tools in diverse crops and livestock make it difficult to develop broad conclusions and best practices, which is further compounded by the diversity of techniques, targets, and processes. Developers and breeders should consult the literature and test as needed to determine which editing technology will be the most effective for their purposes, especially as more tools with altered efficiency and specificity become available. Yet, the lack of off-target edits in studies that employed careful design of targeting components followed by wide testing for on- and off-target edits bodes well for the use of genome editing with proper precautions of target selection and screening.

CRISPR/Cas genome editing is a widely used research technology. Its simplest variant is gene knockout resulting from reparation errors after introduction of dsDNA breaks by Cas nuclease. We compared the outcomes of the break repair by two commonly used nucleases (SpCas9 and LbCas12a) in zebrafish embryos to reveal if application of one nuclease is advantageous in comparison to the other. To address this question, we injected ribonucleoprotein complexes of nucleases and corresponding guide RNAs in zebrafish zygotes and three days later sequenced the target gene regions. We found that LbCas12a breaks resulted in longer deletions and more rare inserts, in comparison to those generated by SpCas9, while the editing efficiencies of both nucleases were the same. On the other hand, overlapping protospacers were shown to lead to similarities in repair outcome, although they were cut by two different nucleases. Thus, our results indicate that the repair outcome depends both on the nuclease mode of action and on protospacer sequence.

We report the analysis of activation by corticosteroids and progesterone of full-length mineralocorticoid receptor (MR) from elephant shark, a cartilaginous fish belonging to the oldest group of jawed vertebrates. Based on their measured activities, aldosterone, cortisol, 11-deoxycorticosterone, corticosterone, 11-deoxcortisol, progesterone and 19-norprogesterone are potential physiological mineralocorticoids. However, aldosterone, the physiological mineralocorticoid in humans and other terrestrial vertebrates, is not found in cartilaginous or ray-finned fishes. Because progesterone is a precursor for corticosteroids that activate elephant shark MR, we propose that progesterone was an ancestral ligand for elephant shark MR. Although progesterone activates ray-finned fish MRs, progesterone does not activate human, amphibian or alligator MRs, suggesting that during the transition to terrestrial vertebrates, progesterone lost the ability to activate the MR. Comparison of RNA-sequence analysis of elephant shark MR with that of human MR suggests that MR expression in the human brain, heart, ovary, testis and other non-epithelial tissues evolved in cartilaginous fishes. Together, these data suggest that progesterone-activated MR may have unappreciated functions in elephant shark ovary and testis.

(1) Background: Hepatoblastoma is the most common primary liver malignancy in children. During its development, metabolic reprogramming becomes particularly relevant due to the liver's intrinsic metabolic functions. Enhanced glycolysis, glutaminolysis, and fatty acid synthesis have been implicated in the proliferation and survival of hepatoblastoma cells. In this study we screened altered over expression of metabolic enzymes in hepatoblastoma tumors at tissue and single cell level. A hepatoblastoma tumor expression metabolic score was established and validated by machine learning; (2) Methods: Starting from Mammalian Metabolic Enzyme Database, bulk RNA-sequencing from GSE104766 dataset was investigated by supervised analyses (tumors versus adjacent liver tissue). Overexpressed enzymes in hepatoblastoma tumors were functionally enriched on KEGG metabolic database to draw a metabolic network with Cytoscape. Activated metabolic markers were used to compute a single-cell metabolic score in human and PDX hepatoblastoma samples from GSE180665 dataset (sc-RNAseq). ROC and area under curve (AUC) were computed on metabolic score. Elasticnet model tuning for metabolic marker expression was performed with r-caret on single cell transcriptome and revealed importance of individual marker to predict tumor cell status; (3) Results: Differential expression analysis on bulk transcriptome identified 287 significantly regulated enzymes between tumor and adjacent liver tissues: 59 of them were found overexpressed in tumors. 45 of the 59 over expressed enzymes were recognized in KEGG database which highlighted a main metabolic network enriched in amino acid metabolism but also carbohydrate, steroid, one carbon, purine, and glucoaminoglycan metabolisms. Based on expression of the 59 over expressed enzymes, the single cell metabolism score computed on GSE180665 dataset allowed to predict tumor cell status with and AUC of 0.98 (sensibility 0.93, specificity: 0.94). Elasticnet model tuned at 0.97 of AUC on individual marker single cell expression ranked top tumor predictive markers by decreasing importance: FKBP10, ATP1A2, NT5DC2, UGT3A2, PYCR1, CKB, GPX7, DNMT3B, GSTP1, and OXCT1; (4) Conclusion: An activated metabolic transcriptional program potentially affecting epigenetic functions was observed by bulk RNAseq in tumors of hepatoblastoma and confirmed at single cell levels in tumor cells.

The methods used to introduce CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas-mediated genome editing into fruit species, as well as the impacts of the application of this technology to activate and knock out of target genes in different fruit trees species including tree development, yield, fruit quality, and tolerance to biotic and abiotic stresses have been firstly described in this review. The application of this gene editing technology could allow the development of new generations of fruit crops with improved traits by targeting different genetic segments or even could facilitate the introduction of traits in elite cultivars without changing other traits. However, at this moment, the scarcity of efficient regeneration and transformation protocols in some species, the fact that many of those procedures are the genotype-dependent or the convenience of segregating the transgenic parts of the CRISPR system represent the main handicaps limiting the potential of genetic editing techniques for fruit trees. Finally, latest news on the legislation and regulations about the use of plants modified through CRISPR/Cas systems has been also discussed.

Preproenkephalin (PPE) is a precursor molecule for multiple endogenous opioid peptides Leu-enkephalin (ENK) and Met-ENK, which are involved in a wide variety of modulatory functions in the nervous system. Despite the functional importance of ENK in the brain, the effect of brain-derived factor(s) on PPE expression is unknown. We report the dual effect of neural epidermal growth factor (EGF)-like-like 2 (NELL2) on PPE gene expression. In cultured NIH3T3 cells, transfection of NELL2 expression vectors induced an inhibition of PPE transcription intracellularly, in parallel with downregulation of protein kinase C signaling pathways and extracellular signal-regulated kinase. Interestingly, these phenomena were reversed when synthetic NELL2 was administered extracellularly. The in vivo disruption of NELL2 synthesis resulted in an increase in PPE mRNA level in the rat brain, suggesting that the inhibitory action of intracellular NELL2 predominates the activation effect of extracellular NELL2 on PPE gene expression in the brain. Biochemical and molecular studies with mutant NELL2 structures further demonstrated the critical role of EGF-like repeat domains in NELL2 for regulation of PPE transcription. These are the first results to reveal the spatio-specific role of NELL2 in the homeostatic regulation of PPE gene expression.

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3-5 base editing windows, 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

The large production of non-degradable petrol-based plastics has become a major global issue due to its environmental pollution. Biopolymers produced by microorganisms such as polyhydroxyalkanoates (PHAs) are gaining potential as a sustainable alternative, but the high cost associated to their industrial production has been a limiting factor. Post-transcriptional regulation is a key step to control gene expression in changing environments and has been reported to play a major role in numerous cellular processes. However, limited reports are available concerning the regulation of PHA accumulation in bacteria, and many essential regulatory factors still need to be identified. Here, we review studies where the synthesis of PHA has been reported to be regulated at the post-transcriptional level, and we analyze the RNA-mediated networks involved. Finally, we discuss the forthcoming research on riboregulation, synthetic and metabolic engineering which could lead to improved strategies for PHAs synthesis in industrial production, thereby reducing the costs currently associated with this procedure.

While the mortality rates of cancer are generally declining, pancreatic cancer persists to be an exception with a 5-year-survival rate of less than 7%. Late diagnosis and resistance to conventional therapies contribute to high mortality rates in spite of the remarkable recent advances in cancer management and research. Consequently, there is an urgent need to find new and unconventional therapeutic targets to improve prognosis and survival of pancreatic cancer patients. In this review, we discuss the transcriptional effects of the most widely used epigenetic inhibitors in pancreatic cancer focusing on Bromodomain and Extraterminal domain (BET) and Histone Deacetylase (HDAC) inhibitors, which are currently highly promising therapeutic options. We suggest that these inhibitors can be better utilized at lower doses which exploit their transcriptional modulatory effects on pancreatic cancer transcriptional programs directed by specific factors such as MYC and FOXA1, rather than simply based on their anti-proliferative effects. This approach can potentially help avoid the intolerable adverse events frequently elicited by the use of these treatments at higher doses. In particular, we underscore the crucial role of distal regulatory elements in mediating the specific effects of these epigenetic inhibitors and propose using them in a more selective and prudent manner.

The symbiotic interaction between leguminous and Bradyrhizobium sp. SUTN9-2 mainly relies on the nodulation process through Nod factors (NFs), while the type IV secretion system (T4SS) acts as an alternative pathway in this symbiosis. Two copies of T4SS (T4SS1 and T4SS2) are located on the chromosome of SUTN9-2. ΔT4SS1 reduces both nodule number and nitrogenase activity in all SUTN9-2 nodulating legumes. The functions of three selected genes (copG1, traG1, and virD21) within the region of the T4SS1 were examined. We generated deleted mutants and tested in Vigna radiata cv. SUT4. ΔtraG1 and ΔvirD21 exhibited lower invasion efficiency at the early stages of root infection but could be lately restored. In contrast, ΔcopG1 completely hindered nodule organogenesis and nitrogenase activity in all tested legumes. ΔcopG1 showed low expression of nodulation gene and ttsI, but exhibited high expression levels of T4SS genes, traG1 and trbE1. The secreted proteins from ΔT4SS1 were down-regulated compared to the wild type. Although ΔcopG1 secreted several proteins after flavonoid induction, T3SS (nopP and nopX) and C4-dicarboxylate transporter (dct) were not detected. These results confirm the crucial role of the copG1 gene as a novel key regulator in the symbiotic relationship between SUTN9-2 and legumes.

Abiotic stresses extensively reduce agricultural crop production globally. Traditional breeding approaches have been widely used to mitigate the risks of abiotic stresses. The discovery of gene editing technology for modifying stress-responsive genes and associated molecular networks has paved the foundation for sustainable crop management against environmental stress. Integrated approaches based on functional genomics and transcriptomics are now expanding the opportunities to elucidate the molecular mechanisms underlying abiotic stress responses. This review summarizes some of the features and weblinks of plant genome databases related to abiotic stress genes utilized for crop improvement. Clustered, regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas) based gene-editing tool has revolutionized stress tolerance research due to its simplicity, versatility, adaptability, flexibility, and broader applications. However, off-target and low cleavage efficiency hinder the successful application of CRISPR/Cas systems. Computational tools have been developed for designing highly competent gRNA with better cleavage efficiency. This powerful genome editing tool offers tremendous crop improvement opportunities, overcoming conventional breeding techniques' shortcomings. Furthermore, we also discuss the mechanistic insights of the CRISPR/Cas9-based genome editing technology. This review summarizes the current advances in understanding plant species' abiotic stress response mechanism and applying the CRISPR/Cas system genome editing technology to develop crop resilience against drought, salinity, temperature, heavy metals, and herbicides.

Advancements in genomics, bioinformatics and genome editing have uncovered new dimensions in gene regulation. Post-transcriptional modifications by the alternative splicing of mRNA transcripts are critical regulatory mechanisms of mammalian gene expression. In the heart, there is an expanding interest in elucidating the role of alternative splicing in transcriptome regulation. Substantial efforts have been directed towards investigating this process in heart development and failure. However, few studies have shed light on alternative splicing products and their dysregulation in congenital heart defects (CHDs). While elegant reports have shown the crucial roles of RNA binding proteins (RBPs) in orchestrating splicing transitions during heart development and failure, the impact of RBPs dysregulation or genetic variation on CHDs has not been fully addressed. Herein, we review the current understanding of alternative splicing and RBPs’ roles in heart development and CHDs and discuss the impacts of perinatal splicing transition and its dysregulation in CHDs. We further summarize discoveries made of causal splicing variants in key transcription factors that have been implicated in CHDs. Improved understanding of the roles of alternative splicing in heart development and CHDs may potentially inform novel preventive and therapeutic advancements for newborn infants with CHDs.

Candida tropicalis, an opportunistic pathogen, ranks among the primary culprits of invasive candidiasis, a condition notorious for its resistance to conventional antifungal drugs. The urgency to combat these drug-resistant infections has spurred the quest for novel therapeutic compounds, with a particular focus on those of natural origin. In this study, we set out to evaluate the impact of Isoespintanol (ISO), a monoterpene derived from Oxandra xylopioides, on the transcriptome of C. tropicalis. Leveraging transcriptomics, our research aimed to unravel the intricate transcriptional changes induced by ISO within this pathogen. Our differential gene expression analysis unveiled 186 differentially expressed genes (DEGs) in response to ISO, with a striking 85% of these genes experiencing upregulation. These findings shed light on the multifaceted nature of ISO's influence on C. tropicalis, spanning a spectrum of physiological, structural, and metabolic adaptations. The upregulated DEGs predominantly pertained to crucial processes, including ergosterol biosynthesis, protein folding, response to DNA damage, cell wall integrity, mitochondrial activity modulation, and cellular responses to organic compounds. Simultaneously, 27 genes were observed to be repressed, affecting functions such as cytoplasmic translation, DNA damage checkpoints, membrane proteins, as well as metabolic pathways like trans-methylation, trans-sulfuration, and trans-propylamine. These results underscore the complexity of ISO's antifungal mechanism, suggesting that it targets multiple vital pathways within C. tropicalis. Such complexity potentially reduces the likelihood of the pathogen developing rapid resistance to ISO, making it an attractive candidate for further exploration as a therapeutic agent. In conclusion, our study provides a comprehensive overview of the transcriptional responses of C. tropicalis to ISO exposure. The identified molecular targets and pathways offer promising avenues for future research and the development of innovative antifungal therapies to combat infections caused by this pathogenic yeast.

Gene or genome editing (GE) revises, removes, or replaces a mutated gene at the DNA level; it is a tool. Gene therapy (GT) offsets mutations by introducing a "normal" version of the gene into the body while the diseased gene remains in the genome; it is a medicine. So far, no in vivo GE product has been approved, as opposed to 22 GT products approved by the FDA. However, several are under development, and the FDA anticipates their fast entry, as evidenced by the FDA's actions of issuing GE-specific guidelines. The potential of GE in treating diseases far supersedes any other modality due to the recognized role of genes in most human diseases. While GT products add missing genes, GE tools can overcome many aberrations. However, GE presents many safety challenges, including off-target impact, delivery consistency, and long-term effects of gene-fixing. However, most of these concerns will be resolved once the FDA begins approving them, leading to hundreds of products capable of treating untreatable diseases.

CRISPR-Cas allows us to introduce desired genome editing, including mutations, epitopes, and deletions with unprecedented efficiency. The development of CRISPR-Cas has progressed to such an extent that it is now applicable in various fields with the help of model organisms. C. elegans is one of the pioneering animals in which numerous CRISPR-Cas strategies have been rapidly es-tablished over the past decade. Ironically, the emergence of numerous methods makes the right choice of method difficult. Choosing an appropriate selection or screening approach is the first step in planning a genome modification. This report summarizes the key features and applications of CRISPR-Cas methods using C. elegans and illustrates key strategies. Our overview of significant advances in CRISPR-Cas will help readers to understand current advances in genome editing and navigate various methods of CRISPR-Cas genome editing.

Adaptive T cell immunotherapy holds great promise for the successful treatment of leukemia as well as other types of cancers. More recently, it was also shown to be an effective treatment option for chronic virus infections in immunosuppressed patients. Autologous or allogeneic T cells used for immunotherapy are usually genetically modified to express novel T cell or chimeric antigen receptors. The production of such cells was significantly simplified with the CRISPR/Cas system allowing deletion or insertion of novel genes at specific locations within the genome. In this review, we describe recent methodological breakthroughs important for the conduction of these genetic modifications, summarize crucial points to be considered when conducting such experiments, and highlight the potential pitfalls of these approaches.

Acute myeloid leukemia (AML) is a hematological cancer with poor outcomes due to a lack of efficacious targeted therapies. The Nuclear Factor of Activated T Cells (NFAT) family of transcription factors is well characterized as a regulator of the cell cycle and differentiation in the myeloid lineage. Recent evidence has demonstrated that NFAT family members may have roles in regulating AML leukemogenesis and resistance to targeted therapy in myeloid leukemias. Furthermore gene expression data from patient samples show that some NFATs are more highly expressed in poorly differentiated AML and after disease relapse, implying that the NFAT family may have roles in specific types of AML. This review outlines the evidence for the role of NFAT in healthy myeloid tissue and explores how NFAT might regulate AML pathogenesis, highlighting the potential to target specific NFAT proteins therapeutically in AML.

There is an urgent need to identify effective therapies for COVID-19. The SARS-CoV-2 host factor protease TMPRSS2 is required for viral entry and thus an attractive target for therapeutic intervention. In mouse, knockout of tmprss2 led to protection against SARS-CoV-1 with no deleterious phenotypes, and in human populations genetic loss of TMPRSS2 does not appear to be selected against. Here, we mined publicly available gene expression data to identify several compounds that down-regulate TMPRSS2. Recognizing the need for immediately available treatment options, we focused on FDA-approved drugs. We found 20 independent studies that implicate estrogenic and androgenic compounds as transcriptional modulators of TMPRSS2, suggesting these classes of drugs may be promising therapeutic candidates for clinical testing and observational studies of COVID-19. We also note that expression of TMPRSS2 is highly variable and skewed in humans, with a minority of individuals having extremely high expression. Combined with literature showing that inhibition of TMPRSS2 protease activity reduces SARS-CoV-2 viral entry in human cells, our results raise the hypothesis that modulation of TMPRSS2 expression is a promising therapeutic avenue for COVID-19.

Post-ovulatory aging (POA) is an inevitable factor during some assisted reproduction technology procedures, and results in poor fertilization rates and impaired embryo development. This study used RNA sequencing analysis and experimental validation to study the similarities and differences between in vivo- and in vitro-matured porcine oocytes before and after POA. Differentially expressed genes (DEGs) between fresh in vivo-matured oocyte (F_vivo) and aged in vivo-matured oocyte(A_vivo) and DEGs between fresh in vitro-matured oocyte (F_vitro) and aged in vitro-matured oocyte(A_vitro) were intersected to explore the co-effects of POA. It was found that "organelles", especially "mitochondria", were significantly enriched GO terms. The expression of genes related to the “electron transport chain” and “cell redox homeostasis” pathways related to mitochondrial function significantly showed low expression patterns in both A_vivo and A_vitro groups. WGCNA analysis to explore gene expression modules specific to A_vivo. Trait-modules association analysis showed that the red modules were most associated with in vivo aging. There are a total of 959 genes in the red module, which are mainly enriched in “RNA binding”, “mRNA metabolic process”, etc. GO terms, and “spliceosome” and “nucleotide excision repair” pathways. DNAJC7, IK, and DDX18 were at the hub of the gene regulatory network. And these genes were all expressed higher in A_vivo. In summary, POA affects the organelle function of oocytes. A_vivo oocytes have some unique gene expression patterns. These genes may be potential anti-aging targets. This study helps us understand the detailed mechanism of POA and provides strategies for researching oocyte aging.

Ovarian cancer is the most frequent cause of deaths in gynecologic malignancies. Many possible mechanisms have been proposed via RNAseq and DNAseq technique recently. However, the driving factors are still obscure. The possible reasons are attributed to the incomplete human reference. This study integrated the canonical mapping-based and mapping-free protocols to extract reliable variations and novel events. We eventually obtained 450 reliable SNVs from the WES data and novel events from the RNAseq data, including 154 SNVs, 462 intron events, two repeats and six splice events. We identified six differentially expressed genes and six contigs that are significantly related to survival prognosis. The recurrent SNVs in significantly differentially expressed genes can be validated in an independent cohort of 20 Chinese ovarian cancer patients.

Detecting gear rim fatigue cracks using vibration signal analysis is often a challenging task, which typically requires a series of signal processing steps to detect and enhance fault features. This task becomes even harder in helicopter planetary gearboxes due to the complex interactions between different gear sets, and the presence of vibration from sources other than the planetary gear set. In this paper, we propose an effectual processing algorithm to isolate and enhance rim crack features and to trend crack growth in planet gears. The algorithm is based on using cepstrum editing (or liftering) of the hunting tooth synchronous averaged signals (angular domain) to extract harmonics and sidebands of the planet gears, and low-pass filtering and minimum entropy deconvolution (MED) to enhance extracted fault features. The algorithm has been successfully applied to a vibration dataset collected from a planet gear rim crack propagation test undertaken in the Helicopter Transmission Test Facility (HTTF) at DSTG Melbourne. In this test, a seeded notch generated by an electric discharge machine (EDM) was used to initiate a fatigue crack that propagated through the gear rim body over 94 load cycles. The proposed algorithm demonstrated a successful isolation of incipient fault features and provided a reliable trending capability to monitor crack progression. Results of a comparative analysis showed that the proposed algorithm outperformed the traditional signal processing approach.

Clustered regularly interspaced short palindromic repeats or CRISPR, one of the major technological tools from nature's toolbox, has revolutionized the scientific world with its potential use in humans and plants. CRISPR Cas9 was first known as an adaptive immune system of bacteria. It is a system that cleaves foreign DNA. It has been exploited to be used as a genome editing tool for correcting genetic diseases in humans, for plants to create stress-resistant plants, and for a variety of different purposes. This review provides a basic overview of its applications in different areas of biological research. It has immense potential for a variety of researches, but it's still a mystery for science. It feels like scientists just know a tip of an iceberg.

Most human genetic disease arises from point mutations. These genetic diseases can theoretically be corrected by gene therapy but clinic practice is still far from mature. Nearly half of the pathogenic single-nucleotide polymorphisms (SNPs) are caused by G:C>A:T or T:A>C:G base changes. The best current methods to repair these changes are by base editing without footprint using recently developed CRISPR-Cas9 technology by David Liu’s lab. These base editing methods have been confirmed with precision and efficiency in cultured mammalian cells, but it is barely confirmed and the efficiency is still very low. Animal models are important in dissecting pathogenic mechanism for human genetic diseases and efficacy testing of base correction in vivo. Cytidine base editor BE4 is a newly developed version of cytidine base editing system that converts cytidine (C) to uridine (U) in cultured mammalian cells but has not been proven in vivo. In this study, we have tested this system in cells to inactivate GFP gene and in mice by introducing single-base substitution that leads to a stop codon in tyrosinase gene. High percentage albino coat-colored mice were obtained from black coat-colored donor zygotes after pronuclei microinjection. Sequencing results showed that expected base changes were obtained with high precision and efficiency (56.25%). There are no off-targeting events identified in predicted off-target sites. Results confirm BE4 system can work in vivo with high precision and efficacy, and has great potentials in clinic to repair human genetic mutations.

Adenosine to inosine (A-to-I) RNA editing is a highly conserved regulatory process carried out by adenosine-deaminases (ADARs) on dsRNAs. Although a considerable fraction of the transcriptome is edited, the function of most editing sites is unknown. Previous studies indicate changes in A-to-I RNA editing frequencies following exposure to several stress types. However, the overall effect of stress on the expression of ADAR targets is not fully understood. Here, we performed high-throughput RNA sequencing of wild-type and ADAR mutant C. elegans worms after heat-shock to analyze the effect of heat-shock stress on the expression pattern of genes. We found that ADAR regulation following heat-shock does not directly involve heat-shock related genes. Our analysis also revealed that lncRNAs and pseudogenes, which have a tendency for secondary RNA structures, are enriched among upregulated genes following heat-shock in ADAR mutant worms. The same group of genes is downregulated in ADAR mutant worms under permissive conditions, which is likely, considering that A-to-I editing protects endogenous dsRNA from RNA-interference (RNAi). Therefore, temperature increases may destabilize dsRNA structures and protect them from RNAi degradation, despite the lack of ADAR function. These findings shed new light on the dynamics of gene expression under heat-shock in relation to ADAR function.

Hepatocellular carcinoma (HCC) is characterized by uncontrolled cellular proliferation driven by aberrant signal transduction. Cells respond to potential carcinogenic signals by inducing cellular senescence (CS) as a defense mechanism. However, assessing CS levels in various tumors is challenging due to heterogeneity and the lack of universal CS markers. This study integrates single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq data from HCC samples to analyze gene expression profiles. HCC-specific senescence-related pathways are identified, and genes from these pathways distinguish molecular subtypes associated with senescence in HCC. These subtypes exhibit significant variations in biological and clinical attributes. A specific CS risk model is developed, revealing associations between CS scores and prognosis, clinical staging, immune infiltration, immunotherapy response, and drug sensitivity. The key gene G6PD is identified as a potential senescence-related target in liver cancer. Molecular experiments confirm the effective suppression of HepG2 cell proliferation, invasion, and migration upon G6PD knockdown. In summary, this analysis enhances our understanding of HCC-specific senescence molecular features and introduces a novel senescence-related biomarker, offering promising implications for future research.

The CRISPR/Cas9 gene editing system has enormous potential in the treatment of multiple genetic disorders. To fully harness this potential, it is essential to deliver CRISPR-Cas9 components safely and effectively into the nuclei of target cells. In this study, we tested the amphipathic cell-penetrating peptide LAH5, previously used for DNA delivery, to intracellularly deliver spCas9: sgRNA ribonucleoprotein (RNP) and RNP/single-stranded homology-directed repair (HDR) template. We discovered that the LAH5 peptide effectively formed nanocomplexes with both RNP and RNP/HDR cargo, and that these nanocomplexes were taken up by cells effectively. Peptide/RNP nanocomplexes were characterized for size and charge, and nanocomplexation with RNP and RNP/HDR cargo was demonstrated via an electrophoretic mobility shift assay. Various ratios of peptide/RNP nanocomplexes were functionally screened on fluorescent reporter cell lines for gene editing and HDR-mediated gene correction. Additionally, targeted gene editing of the CCR5 gene was demonstrated in various cell lines. This LAH5-based delivery strategy paves the way for the development of therapeutic delivery systems for CRISPR/Cas-based genomic engineering.

With the rapid population growth, there is an urgent need for innovative crop improvement approaches to meet the increasing demand for food. Classical crop improvement approaches involve, however, a backbreaking process that cannot equipoise with increasing crop demand. RNA based approaches i.e. RNAi-mediated gene regulation and site-specific nuclease based CRISPR/Cas9 system for gene editing has made advances in the efficient targeted modification in many crops for the higher yield and resistance to diseases and different stresses. In functional genomics, RNA interference (RNAi) is a propitious gene regulatory approach that plays a significant role in crop improvement by permitting down-regulation of gene expression by small molecules of interfering RNA without affecting the expression of other genes. Gene editing technologies viz. clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (CRISPR/Cas) have appeared prominently as a powerful tool for precise targeted modification of nearly all crops genome sequence to generate variation and accelerate breeding efforts. In this regard, the review highlights the diverse roles and applications of RNAi and CRISPR/Cas9 system as powerful technologies to improve agronomically important plants to enhance crop yields and increase tolerance to environmental stress (biotic or abiotic). Ultimately, these technologies can prove to be important in view of global food security and sustainable agriculture.

Sickle cell disease is characterized by stiff, “sickled” red blood cells that have difficulty moving through the bloodstream and do not efficiently carry oxygen. It is an inherited disease with severely limited treatment options, and is caused by a point mutation. Its prevalence in black and brown communities makes the already limited treatment options even less accessible. Base editing and prime editing are two relatively recent discoveries in the field of genome editing and were developed after the groundbreaking discovery of the CRISPR Cas9 system. While not fully tested, they hold a lot of promise in providing alternative treatment options for sickle cell disease. Both editing systems are able to install individual point mutations in the beta globin gene, which is where the sickle cell mutation occurs, and can thus cure sickle cell disease (in theory). In this paper we outline the mechanisms of CRISPR-Cas9 systems and base and prime editing, and provide insight into how to apply them to treat SCD. Further investigation should be done on specific editing systems and designs to use to ensure optimal treatment of SCD.

An analysis of 1300+ existing ACE mutations revealed that 400+ are damaging and led us to hy-pothesize that carriers of heterozygous loss-of-function (LoF) ACE mutations (which result in low ACE levels) could be at risk for the development of late-onset Alzheimer’s disease (AD) [Danilov, 2024]. Here we quantified blood ACE levels in EDTA-plasma from 41patients with 10 different heterozygous ACE mutations, as well as 33 controls, and estimated the effect of these mutations on ACE phenotype using a set of mAbs to ACE and two ACE substrates. We found that relatively frequent (~1%) AD-associated ACE mutations in the N domain of ACE, Y215C and G325R are truly damaging and, likely, transport-deficient, with ACE levels in plasma only ~50% of controls. Another AD-associated ACE mutation, R1250Q, in the cytoplasmic tail, did not cause a decrease in ACE and, likely, did not affect surface ACE expression. We have also developed a method to identify patients with anti-catalytic mutations in the N domain. These mutations may result in reduced degradation of amyloid beta peptide Aβ42, an important component for amyloid deposition. Consequently, these could pose a risk factor for the development of AD. Therefore, a systematic analysis of blood ACE levels in patients with all ACE mutations has potential to identify individuals at an increased risk of late-onset AD. These individuals may benefit from future preventive or therapeutic interventions involving a combination of chemical and pharmacological chaperones, as well as proteasome inhibitors, aiming to enhance ACE protein traffic. This approach has been previously demonstrated in a cell model of the transport-deficient ACE mutation, Q1069R [Danilov, 2010].

DIPA-CRISPR is a revolutionary gene editing technology that has set new benchmarks in precision and efficacy. It builds upon the CRISPR-Cas9 system, introducing unparalleled specificity and minimizing unintended effects. Its potential applications in addressing complex genetic disorders offer hope for more effective disease management. The integration of DIPA-CRISPR into gene therapy research signifies a monumental leap forward in medical science and biotechnology. This article explores the DIPA-CRISPR methodology, its core principles, and recent technological strides in refining the technique. It highlights the implications of these developments on the treatment of genetic conditions and discusses future prospects and innovations within the field. The transformative potential of DIPA-CRISPR in disease management and the broader spectrum of genetic engineering is underscored, heralding a new age of medical and scientific discovery. The emphasis on addressing both technical and ethical challenges underscores the complexity and responsibility that accompanies such powerful technological advancements. The future prospects of DIPA-CRISPR, from pest management to combating global infectious diseases and transforming oncology, underscore the need for continued innovation coupled with rigorous ethical oversight.

Coordination of cellular biological processes is regulated in part via metabolic enzymes acting to match cellular metabolism to current conditions. The acetate activating enzyme, acyl-coenzyme A synthetase short-chain family member 2 (Acss2) has long been considered to have a predominantly lipogenic function. More recent evidence suggests that this enzyme has regulatory functions in addition to its role in providing acetyl-CoA for lipid synthesis. We used Acss2 knockout mice (Acss2-/-) to further investigate the roles this enzyme plays in three physiologically distinct organ systems that make extensive use of lipid synthesis and storage including the liver, brain and adipose tissue. We examined the resulting transcriptomic changes resulting from Acss2 deletion and assessed these changes in relation to fatty acid constitution. We find that loss of Acss2 leads to dysregulation of numerous canonical signaling pathways, upstream transcriptional regulatory molecules, cellular processes, and biological functions, which were distinct in the liver, brain and mesenteric adipose tissues. The detected organ-specific transcriptional regulatory patterns reflect the complementary functional roles of these organ systems within the context of systemic physiology. While alterations in transcriptional states were evident, the loss of Acss2 resulted in few changes in fatty acid constitution in all three organ systems. Overall, we demonstrate Acss2 loss institutes organ-specific transcriptional regulatory patterns reflecting the complementary functional roles of these organ systems. Collectively, these findings provide further confirmation that Acss2 regulates key transcription factors and pathways under well-fed, non-stressed conditions, and acts as a transcriptional regulatory enzyme.

Due to an increase in the consumption of food, feed, and fuel and to meet global food security needs for the rapidly growing human population, there is a necessity to obtain high-yielding crops that can adapt to future climate changes. Currently, the main feed source used for ruminant livestock production is forage grasses. In temperate climate zones, perennial grasses grown for feed are widely distributed and tend to suffer under unfavorable environmental conditions. Gene editing has been shown to be an effective tool for the development of abiotic stress-resistant plants. The highly versatile CRISPR-Cas system enables increasingly complex modifications in genomes while maintaining precision and low off-target frequency mutations. In this review, we provide an overview of forage grass species that have been subjected to gene editing. We offer a perspective view on the generation of plants resilient to abiotic stresses. Due to the broad factors contributing to these stresses the review focuses on drought, salt, heat, and cold stresses. The application of new genomic techniques (e.g., CRISPR-Cas) allows addressing several challenges caused by climate change and abiotic stresses for developing forage grass cultivars with improved adaptation to the future climatic conditions. Gene editing will contribute towards developing safe and sustainable food systems.

Global crop yield and food security are being threatened by phytophagous insects. Innovative methods are required to increase agricultural output while reducing reliance on hazardous synthetic insecticides. It appears to be quite effective at reducing production costs and boosting farm profitability to use the ground-breaking CRISPR-Cas technology to create plants that are insect resistant. In contrast, this new technique can modify an insect's genome to either produce gene drive or get beyond an insect's tolerance to various insecticides. This paper reviews and critically discusses the use of CRISPR-Cas genome editing technology in long-term insect pest management. The emphasis of this review is on the prospective uses of the CRISPR-Cas system for insect stress management in crop production by creating genome-edited crops and insects. The potential and difficulties of using CRISPR-Cas technology to reduce pest stress in crop plants are critically examined and discussed.

Background: Cardiac development is a complex developmental process. The early cardiac straight tube is composed of an external myocardial layer and an internal endocardial lining. Soon after rightward looping, the embryonic heart becomes externally covered by a new epithelial lining, the embryonic epicardium. A subset of these embryonic epicardial cells migrate and colonize the embryonic myocardium, contributing to distinct cell types. In recent years, our understanding of the molecular mechanisms that govern proepicardium and embryonic epicardium formation has greatly increased. We have recently witnessed a novel layer of complexity governing gene regulation with the discovery of non-coding RNAs. Our laboratory recently identified three distinct lncRNAs, adjacent to the Wt1, Bmp4 and Fgf8 chicken gene loci, with enhanced expression in the proepicardium that are distinctly regulated by Bmp, Fgf and thymosin 4, providing support of their plausible implication in epicardial formation. Methods: Expression of lncRNAs was analyzed in different chicken and mouse tissues as well as their subcellular distribution in chicken proepicardial, epicardial, ventricle explants and in different murine cardiac cell types. lncRNA transcriptional regulation was analyzed by using siRNAs and expression vectors of different transcription factors in chicken and mouse. Antisense oligonucleotides were used to inhibit Gm14014 expression. Cardiac injury was induced ex vivo in mouse ventricle explants by cryoinjury. Furthermore, RT-qPCR, immunocytochemistry, RNA pulldown, Western blot, viability and cell migration assays were conducted to investigate the biological function of Wt1_76127 and Gm14014. Results: We demonstrated that Wt1_76127 in chicken and its evolutionarily conserved homologue Gm14014 in mice are widely distributed in different embryonic and adult tissues and distinctly regulated by cardiac enriched transcription factors, particularly Mef2c and Nkx2.5. Gm14014 is distinctly regulated in mouse ventricular cryoinjury ex vivo models, displaying a negative correlation with epicardial and epithelial to mesenchymal transition markers. Furthermore, silencing assays demonstrated that mouse Gm14014, but not chicken Wt1_76127, is essential for epicardial, but not endocardial or myocardial cell migration. Such process is governed by partnering with Myl9, promoting cytoskeletal remodeling. Conclusion: Our data evidence that Gm14014 plays a pivotal role in epicardial cell migration essential for heart regeneration.

To address the response of small RNAs (sRNAs) biogenesis to drought stress, we identified sRNAs biogenesis genes, including 11 encoding argonauts (AGO), 8 encoding dicer-like proteins (DCL), and 9 encoding RNA-dependent RNA polymerases (RDR) in the peach genome. Notably, the largest numbers of sRNAs biogenesis genes are located to chromosome 1. The PAZ, PIWI, and MID domains were identified in PpAGOs, while the ribonuclease IIIa and IIIb domains were characterized in PpDCLs. The RDRP domain was recognized in PpRDRs. Orthologous similarity and collinearity analyses between Arabidopsis and peach revealed 5, 1, and 2 collinear blocks in AGOs, DCLs, and RDRs, respectively. Moreover, 41, 40, and 42 cis-acting elements were located in the promoters of PpAGOs, PpDCLs, and PpRDRs, respectively, with the majority related to drought stress response. Analysis of RNA sequencing (RNA-seq) data revealed that sRNAs biogenesis genes were involved in drought stress response in different tissues. Furthermore, the expression of candidate genes was verified in two peach cultivars, Beijing 2-7 (BJ2-7) and Sinai (SN), which are drought tolerance and sensitive, respectively. Interestingly, the expression of PpAGO2b, PpDCL2b, PpDCL4, and PpRDR4 genes was induced in ‘BJ2-7’ but inhibited in ‘SN’ under drought stress. Overall, this study provides insight into the roles of sRNA biogenesis genes in response to drought stress in peach.

Chimeric antigen receptor (CAR) T-cell therapy has greatly improved outcomes for patients with relapsed or refractory hematological malignancies. However, challenges such as treatment resistance, relapse, and severe toxicity still hinder its widespread clinical application. Traditional transcriptome analysis has provided limited insights into the complex transcriptional landscape of both leukemia cells and engineered CAR-T cells, as well as their interactions within the tumor microenvironment. However, with the advent of single-cell sequencing techniques, a paradigm shift has occurred, providing robust tools to unravel the complexities of these factors. These techniques enable unbiased analysis of cellular heterogeneity and molecular patterns. These insights are invaluable for precise receptor design, guiding gene-based T cell modification, and optimizing manufacturing conditions. Consequently, this review utilizes modern single-cell sequencing techniques to clarify the transcriptional intricacies of leukemia cells and CAR-Ts. The aim of this manuscript is to discuss the potential mechanisms that contribute to the clinical failures of CAR-T immunotherapy. We will examine the biological characteristics of CAR-Ts, the mechanisms that govern clinical responses, and the intricacies of adverse events. By exploring these aspects, we hope to gain a deeper understanding of CAR-T therapy, which will ultimately lead to improved clinical outcomes and broader therapeutic applications.

Sweet sorghum is an important bioenergy grass and valuable forage with a strong adaptability to saline environments. However, little is known about the mechanisms of sweet sorghum coping with ion toxicity under salt stresses. Here, we first evaluated the salt tolerance of a sweet sorghum cultivar “Lvjuren” and determined its ion accumulation traits under NaCl treatments; then explored key genes involved in Na+, Cl−, K+ and NO3− transport using transcriptome profiling and qRT-PCR method. The results showed that the growth and photosynthesis of sweet sorghum were unaffected by 50 and 100 mM NaCl treatments, indicative of a strong tolerance of this species to salt stresses. Under NaCl treatments, sweet sorghum could efficiently exclude Na+ from shoots and accumulate Cl− in leaf sheaths to avoid their overaccumulation in leaf blades; meanwhile, it possessed a prominent ability to sustain NO3− homeostasis in leaf blades. Transcriptome profiling identified several differentially expressed genes associated with Na+, Cl−, K+ and NO3− transport in roots, leaf sheaths and leaf blades of sweet sorghum after 200 mM NaCl treatment for 6 and 48 h. Moreover, transcriptome data and qRT-PCR results indicated that HKT1;5, CLCc and NPF7.3-1 should be key genes involved in Na+ retention in roots, Cl− accumulation in leaf sheaths and maintenance of NO3− homeostasis in leaf blades, respectively. Many TFs were also identified after NaCl treatment, which should play important regulatory roles in salt tolerance of sweet sorghum. This work lays a preliminary foundation for clarifying the molecular basis underlying the adaptation of sweet sorghum to adverse environments.

Aedes aegypti (L.) is the primary vector of chikungunya, dengue, yellow fever and Zika viruses. The leucine-rich repeats (LRR)-containing domain is evolutionarily conserved in many proteins associated with innate immunity in invertebrates and vertebrates, as well as plants. We focused on the AaeLRIM1 and AaeAPL1 gene expressions in response to Zika virus (ZIKV) and Chikungunya virus (CHIKV) infection using a time course study, as well as the developmental expressions in the eggs, larvae, pupae, and adults. RNA-seq analysis data provided 60 leucine-rich repeat related transcriptions in Ae. aegypti in response to Zika virus (Accession number: GSE118858, https://www.ncbi.nlm.nih.gov/gds/?term=GSE118858). RNA-seq analysis data showed that AaeLRIM1 (AAEL012086-RA) and AaeAPL1 (AAEL009520-RA) were significantly upregulated 2.5 and 3-fold during infection by ZIKV 7-days post infection (dpi) of an Ae. aegypti Key West strain compared to an Orlando strain. The qPCR data showed that LRR-containing proteins AaeLRIM1, AaeAPL1 and five paralogues were expressed 100-fold lower than other nuclear genes, such as defensin, during all developmental stages examined. Together, these data provide insights into transcription profiles of LRR proteins of Ae. aegypti during its development and in response to infection with emergent arboviruses.

This mini-review provides an overview of traditional, emerging, and future applications of lactic acid bacteria (LAB) and discusses how genome editing tools can be used to overcome current challenges in all these applications. It also describes currently available tools and how these can be further developed, and takes current legislation into account. Genome editing tools are necessary for the construction of strains for new applications and products, but can also play a crucial role in traditional ones, such as food and probiotics, as a research tool for understanding mechanistic insights and discovering new properties. Traditionally, recombinant DNA techniques for LAB have strongly focused on being food-grade, but they lack throughput and the number of genetically tractable strains is still rather limited. Further tool development in this direction will enable rapid construction of multiple mutants or mutant libraries on a genomic level in a wide variety of LAB strains. We also propose an iterative Design-Build-Test-Learn workflow cycle for LAB cell factory development based on systems biology, with “cell factory” expanding beyond its traditional meaning of production strains and making use of high-throughput genome editing tools to advance LAB understanding, applications and strain development.

Coronaviruses constitute a global threat to human and animal health. It is essential to investigate the long-distance RNA-RNA interactions that approximate remote regulatory elements in strategies, including genome circularization, discontinuous transcription, and transcriptional enhancers, aimed at rapid replication of their large genomes, pathogenicity, and immune evasion. Based on the primary sequences and modeled RNA-RNA interactions of two experimentally de-fined coronaviral enhancers, we detected via in silico primary and secondary structural analysis potential enhancers in various coronaviruses, from the phylogenetically ancient avian IBV to the recently emerged SARS-CoV-2. These potential enhancers possess a core duplex-forming region that could transition between closed and open states, as molecular switches directed by viral or host factors. The open state would permit the enhancer to pair with remote sequences in the viral genome and modulate the expression of crucial genes involved in viral replication and host immune evasion. Consistently, variations in the predicted IBV enhancer region or its distant targets coincide with cases of viral attenuation possibly driven by decreased expression of the ORF3a immune evasion protein. The annotated enhancer sequences could inform structural prediction tools and antiviral interventions if validated experimentally.

Jujube (Ziziphus jujuba Mill.) stands as a pivotal fruit tree with significant economic, ecological, and social value. Recent years have witnessed remarkable strides in multi-omics-based biological re-search on jujube. This review began by summarizing advancements in jujube genomics. Subse-quently, we provided a comprehensive overview of the integrated application of genomics, tran-scriptomics, and metabolomics to explore pivotal genes governing jujube domestication traits, quality attributes (including sugar synthesis, terpenoids, and flavonoids), and responses to abiotic stress, and discussed the transcriptional regulatory mechanisms underlying these traits. Further-more, challenges in multi-omics research on jujube biological traits were outlined, and we pro-posed the integration of resources such as pan-genomics and sRNAome to unearth key molecules and regulatory networks influencing diverse biological traits. Incorporating these molecules into practical breeding strategies, including gene editing, transgenic approaches, and progressive breeding, holds the potential for achieving molecular-design breeding and efficient genetic en-hancement of jujube.

The diversity of leaf characteristics, including leaf color, is a prominent subject of study in plant science. Leaf color is predominantly determined by the synthesis and functionality of chlorophyll, a key component of photosynthesis. The regulation of chlorophyll synthesis and degradation involves complex gene interactions, and disruptions in these processes can lead to abnormal chlorophyll synthesis and impact leaf color. This study focuses on Bambusa multiplex f. silverstripe, a natural variant with various leaf colors, including green, white, and green-white leaves. The variations in leaf color are attributed to genetic factors and their influence on gene expression. By employing RNA-seq, we investigate the molecular mechanisms behind chlorophyll anomalies and genetic factors in Silverstripe. Our findings shed light on the complexity of gene interactions and regulatory networks that underlie leaf color diversity and provide valuable insights for future research and plant breeding.

An increasing amount of evidence indicates the critical role of the cutaneous nervous system in the initiation and maintenance of psoriatic skin lesions by neurogenic inflammation. However, molecular mechanisms affecting cutaneous neurons are largely uncharacterized. Therefore, we reanalyzed a psoriatic RNA sequencing dataset from published transcriptome experiments of nearly 300 individuals. Using the Ingenuity Pathway Analysis software, we associated several hundreds of differentially expressed transcripts (DETs) to nervous system development and functions. Since neuronal projections were previously reported to be affected in psoriasis, we performed an in-depth analysis of neurite formation-related processed. Our in silico analysis suggests that SEMA-PLXN and ROBO-DCC-UNC5 regulating axonal growth and repulsion are differentially affected in non-lesional and lesional skin samples. We identified opposing expressional alterations in secreted ligands for axonal guidance signaling (RTN4/NOGOA, NTNs, SEMAs, SLITs) and non-conventional axon guidance regulating ligands, including WNT5A and their receptors, modulating axon formation. These differences in neuritogenesis may explain the abnormal cutaneous nerve filament formation described in psoriatic skin. The processes also influence T cell activation and infiltration, thus highlighting an additional angle of the crosstalk between the cutaneous nervous system and the immune responses in psoriasis pathogenesis, in addition to the known neurogenic pro-inflammatory mediators.

CRISPR-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach and it can generate unintended transgenic integrations, while Cas9 prolonged expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of treated cells, especially plant cells, is still challenging. These downfalls can be avoided with the delivery of preassembled ribonucleoprotein complexes (RNPs) composed of purified recombinant enzyme Cas9 and in vitro- transcribed guide RNA (gRNA) molecules in a protoplast system. We therefore aimed to develop the first DNA-free protocol for gene-editing in maize and introduced RNPs into their protoplasts with PEG 4000. We performed effective transformation of maize protoplasts using different gRNAs sequences targeting the inositol phosphate kinase gene and applying two different exposure times to RNPs. Using low-cost Sanger sequencing protocol, we observed an efficiency rate of 0.85 up to 5.85%, which is equivalent to DNA-free protocols used in other plant species. A positive correlation was displayed between exposure time and mutation frequency. Mutation frequency was gRNA sequence- and exposure time-dependent. In summary, we demonstrated the suitability of RNP transfection as an effective screening platform for gene-editing in maize. This efficient and relatively easy assay method for selection of gRNA suitable for editing of gene of interest will be highly useful for genome editing in maize, since genome size and GC-content are large and high in maize genome, respectively. Nevertheless, the large amplitude of mutations at target site requires scrutiny when checking mutations at off-target sites and potential safety concerns.

Rapid progress in gene editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) has revolutionized the study of gene and genome function and genetic disease correction. While numerous genetically modified cellular and animal models have been created to understand biological processes, the clinical application of CRISPR/Cas tools has been impeded by off-targeting and delivery problems. It is generally accepted that the delivery of CRISPR in the form of a ribonucleoprotein complex (RNP) substantially reduces the time of DNA exposure to the effector nuclease, minimizing off-target effects and facilitating clinical usage. This review focuses on CRISPR/Cas RNP delivery with retro/lentiviral particles and exosomes, whose parallel production by cells transfected with viral vectors is underestimated. We critically evaluate specific mechanisms of extracellular particle formation and loading with CRISPR/Cas for each system. Additionally, the details of Cas-nanoparticle entry and uncoating, previously unappreciated in the context of gene editing efficiency, are discussed. Based on existing knowledge about the consequences of intervention in retroviral assembly, entry, or exosome formation, we outline the potential problems with CRISPR/Cas delivery using extracellular nanoparticles and ways to address them.

The transcriptional response of human blood leukocytes to SARS-CoV-2 infection was investigated focusing on the differences between mild and severe cases and between age subgroups. Weighted gene co-expression network analysis and comparative gene expression analysis were used. Three transcriptional modules positively associated with the traits of interest and their respective high hierarchy genes were identified. Enrichment analyses showed that the yellow module, associated with severe cases and older patients, had an overrepresentation of genes involved in inflammatory and innate immune responses, and neutrophil activation. The magenta and black modules, associated with disease severity and younger patients, contained genes related to innate immunity and inflammation and genes that regulate those responses. Subnetworks for these modules were constructed using genes enriched for innate immunity, inflammation, immunoregulation and differentially expressed genes (severe vs. mild). Their analysis evidenced that immunoregulatory functions are more activated in the modules associated with younger patients, what may help to explain the better disease course and faster recovery observed in younger COVID-19 patients. Comparative gene expression analysis between severe and mild groups, followed by gene enrichment and normalized gene expression analyses, revelated a set of 23 potential biomarkers for COVID-19 severity, of which 13 are newly described.

Abiotic stresses, including drought, salinity, cold, heat, and heavy metals, extensively reduce global agricultural production. Approaches such as conventional breeding and transgenic breeding have been widely used to cope with these environmental stresses. The clustered regularly interspaced short palindromic repeat- Cas (CRISPR/Cas) based gene-editing tool has revolutionized due to its simplicity, accessibility, adaptability, flexibility, and wide applicability. This system has a great potential to build up crop varieties with enhanced tolerance against abiotic stresses. In this review, we summarize the most recent findings on understanding the mechanism of abiotic stress response in plants and the application of CRISPR/Cas mediated gene-editing system towards enhanced tolerance to drought, salinity, cold, heat, and heavy metals stresses. Furthermore, in this review, we highlighted the recent advancements in prime editing and base editing tools for crop improvement.

The major threats to the sustainable supply of forest tree products are adverse climate, pests and diseases. Climate change, exemplified by increased drought, poses a unique threat to global forest health. This is attributed to the unpredictable behavior of forest pathosystems, which can favor fungal pathogens over the host under persistent drought stress conditions in the future. Currently, the effects of drought on tree resistance against pathogens are hypothetical, thus research is needed to identify these correlations. Norway spruce (Picea abies) is one of the most economically important tree species in Europe, and is considered highly vulnerable to changes in climate. Dedicated experiments to investigate how disturbances will affect the Norway spruce - Heterobasidion sp. pathosystem are important, in order to develop different strategies to limit the spread of H. annosum s.l. under the predicted climate change. Here, we report a transcriptional study to compare Norway spruce gene expressions to evaluate the effects of water availability and the infection of Heterobasidion parviporum. We performed inoculation studies of three-year-old saplings in a greenhouse (purchased from a nursery). Norway spruce saplings were treated in either high (+) or low (-) water groups: high water group received double the water amount than the low water group. RNA was extracted and sequenced. Similarly, we quantified gene expression levels of candidate genes in biotic stress and jasmonic acid (JA) signaling pathways using qRT-PCR, through which we discovered a unique preferential defense response of H. parviporum-infected Norway spruce under drought stress at the molecular level. Disturbances related to water availability, especially low water conditions can have negative effects on the tree host and benefit the infection ability of the pathogens in the host. From our RNA-seq analysis, 114 differentially expressed gene regions were identified between high (+) and low (-) water groups under pathogen attack. None of these gene pathways were identified to be differentially expressed from both non-treated and mock-control treatments between high (+) and low (-) water groups. Finally, only four genes were found to be associated with drought in all treatments.

All RNA viruses deliver their genomes into target host cells through processes distinct from normal trafficking of cellular RNA transcripts. The delivery of viral RNA into most cells hence triggers innate antiviral defenses that recognize viral RNA as foreign. In turn, viruses have evolved mechanisms to subvert these defenses, allowing them to thrive in target cells. Therefore, drugs activating defense to exogenous RNA could serve as broad-spectrum antiviral drugs. Here we show that transcriptional signatures associated with cellular responses to the delivery of a non-viral exogenous RNA sequence into human cells predict small molecules with broad-spectrum antiviral activity. In particular, transcriptional responses to the delivery of Cas9 mRNA into human hematopoietic stem and progenitor cells (HSPCs) highly matches those triggered by small molecules with broad-spectrum antiviral activity such as emetine, homoharringtonine, pyrvinium pamoate and anisomycin, indicating that these drugs are potentially active against other RNA viruses. Furthermore, these drugs have been approved for other indications and could thereby be repurposed to novel viruses. We propose that the antiviral activity of these drugs to SARS-CoV-2 should therefore be determined as they have been shown as active against other coronaviruses including SARS-CoV-1 and MERS-CoV. Indeed, two of these drugs- emetine and homoharringtonine- were independently shown to inhibit SARS-CoV-2 as this article was in preparation. These drugs could also be explored as potential adjuvants to COVID-19 vaccines in development due to their potential effect on the innate antiviral defenses that could bolster adaptive immunity when delivered alongside vaccine antigens.

The advent of CRISPR/Cas9 technology has revolutionized genome editing, enabling the attainment of once-unimaginable goals. CRISPR/Cas's groundbreaking attributes lie in its simplicity, versatility, universality, and its independence from customized DNA-protein systems, erasing the need for specialized expertise and broadening its scope of applications. Beyond its editing prowess, the discovery of novel Cas-based systems has spawned an array of additional biotechnological tools, empowering both fundamental and applied research. Researchers have harnessed CRISPR/Cas for gene regulation, deepening insights into gene expression and epigenetic changes. Precisely targeting DNA or RNA sequences, CRISPR/Cas effectively identifies and counters pathogens. Furthermore, it aids in genome imaging and sequencing, revealing genome spatial organization and chromatin dynamics. The non-editing applications of CRISPR/Cas exhibit tremendous potential across diverse domains, including diagnostics, biotechnology, and fundamental research. This article reviews and critically evaluates the primary CRISPR/Cas-based tools developed for plants and animals, underlining their transformative impact.

A single paragraph of about 200 words maximum. For research articles, abstracts should give a pertinent overview of the work. We strongly encourage authors to use the following style of structured abstracts, but without headings: (1) Background: Place the question addressed in a broad context and highlight the purpose of the study; (2) Methods: briefly describe the main methods or treatments applied; (3) Results: summarize the article’s main findings; (4) Conclusions: indicate the main conclusions or interpretations. The abstract should be an objective representation of the article and it must not contain results that are not presented and substantiated in the main text and should not exaggerate the main conclusions.

With the continuous update of gene editing technology, great progress has been made in livestock breeding, researchers have produced gene edited pigs, cattle, sheep and other improved livestock. Gene editing technology is a genetic means to knock in, knock out, delete, inhibit, activate or replace specific bases of DNA or RNA sequences at the genome level to achieve accurate modification, and can edit genes at a fixed point without the need for DNA templates. In recent years, although regular clusters of interspaced short palindromic repeats (CRISPR)/Cas9 system mediated gene editing technology has been widely used in animal genetic breeding research, but so far, the system's precise insertion efficiency of foreign genes is not high enough, and there are certain off-target effects, and it is not enough to be used in genome editing of large livestock such as cashmere goats. In this paper, the development status, challenges, application prospects and look into the distance of CRISPR/ Cas9-mediated precisiongene editing technology in livestock breeding were reviewed, in order to provide theoretical reference for livestock gene function analysis, genetic improvement and livestock breeding with local economic characteristics.

The indigenous communities across the globe especially in the rural areas consume locally available plants known as Traditional Food Plants (TFPs) for their nutritional and health-related needs. Recent research shows that many of the traditional food plants are highly nutritious as they contain health beneficial metabolites, vitamins, mineral elements and other nutrients. Excessive reliance on the mainstream staple crops has its own disadvantages. TFPs are nowadays considered important crops of the future and can act as supplementary foods for the burgeoning global population. They can also act as emergency foods in times of pandemics and other situations like COVID-19. The current situation necessitates locally available alternative nutritious TFPs for sustainable food production. To increase the cultivation or improve the traits in TFPs, it is essential to understand the molecular basis of the genes that regulate some important traits such as nutritional components and resilience to biotic and abiotic stresses. The integrated use of modern omics and gene editing technologies provide great opportunities to better understand the genetic and molecular basis of superior nutrient content, climate-resilient traits and adaptation to local agroclimatic zones. Recently, realising the importance and benefits of TFPs, scientists have shown interest in the prospection and sequencing of traditional food plants for their improvements, further cultivation and mainstreaming. Integrated omics such as genomics, transcriptomics, proteomics, metabolomics and ionomics are successfully used in plants and have provided a comprehensive understanding of gene-protein-metabolite networks. Combined use of omics and editing tools has led to successful editing of beneficial traits in few TFPs. This suggests that there is ample scope of integrated use of modern omics and editing tools/techniques for improvement of TFPs and their use for sustainable food production. In this article, we highlight the importance, scope and progress towards improvement of TFPs for valuable traits by integrated use of omics and gene editing techniques.

Several genetic tools have been developed for Clostridium acetobutylicum utilizing 5-fluorouracil (5FU) or 5-fluorocytosine (5FC) resistance as a selection method. A method based on the integration, by single crossing-over, of a suicide plasmid (pCat-upp) followed by selection for the second crossing-over using a counter-selectable marker (the upp gene and 5FU resistance) was recently developed for genome editing in C. acetobutylicum. This method allows the modification of the genome without any marker or scar left in a strain of C. acetobutylicum that is ∆upp. Unfortunately, 5FU has strong mutagenic properties inducing mutations in the strain’s genome. After numerous applications of the pCat-upp/5FU system for genome modification in C. acetobutylicum, the bacteria became completely resistant to 5FU in the presence of the upp gene, resulting in failure in the selection for the second crossing-over. It was found that the potential repressor of the pyrimidine operon, PyrR, was mutated at position A115, leading to the 5FU resistance of the strain. We developed two plasmids, one overexpressing the native pyrR gene and a suicide plasmid carrying a non-mutated and optimized pyrR gene (pyrR*) and upp, which allowed us to restore the 5FU sensitivity of the strain. We also improved the pCat-upp/5FU system by reducing the concentration of 5FU from 1mM to 5 µM using a define synthetic medium.

Fruit production is an important part of the gross domestic product for many countries around the world especially to those who have a strong focus on agriculture. However, long-term maintenance and yield stability of fruit production may be threatened by the ongoing climate change and its consequences like extended drought periods, heavy rain events, and floodings. Genome editing, with its progressive technological developments, offers opportunities to adapt relevant fruit plant species to new climatic conditions. Among modern genome editing techniques, CRISPR/Cas, in particular, has the potential to support breeding for those fruit plant species with extended breeding cycles, e.g., perennial fruits. In this review, we discuss CRISPR/Cas and other genome editing techniques in detail and how these techniques can be applied to support the breeding of fruit plant species for adaptation to changing climates. The chronological history of CRISPR/Cas9 systems, their associated computational tools, genomic data sources, transformation methods along with their delivery vehicles, quality improvement, environmental-stress resiliency, limitations, and future perspectives will also be discussed with respect to securing future global fruit production.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus considered as a threat to human health due to large epidemics and serious clinical outcomes such as microcephaly in newborns. Like all flaviviruses, ZIKV relies on the cellular machinery to complete its viral cycle, with the endoplasmic reticulum (ER) being the critical site of viral replication factories. The sudden high protein load in the ER induces an ER stress to which the cell responds with an appropriate unfolded protein response (UPR) in an attempt to restore its disturbed homeostasis. When the restoration fails, the cell signaling leads to a programmed cell death by apoptosis with the upregulation of the UPR-induced C/EBP homologous protein (CHOP) which acts as the main trigger for this fatal outcome. Our previous studies have shown the ability of ZIKV to manipulate various cellular responses in order to optimize virus production. ZIKV is able to delay apoptosis to its benefit and although ER stress is induced, the UPR is not complete. Here we discovered that ZIKV impairs the expression of CHOP/DDIT3, the main factor responsible of ER-stress driven apoptosis. Surprisingly, the mechanism does not take place at the transcriptional level but at the translational level.

Gene expression profiling data contains more information than is routinely extracted with standard approaches. Here we present Fold-change-Specific Enrichment Analysis (FSEA), a new method for functional annotation of differentially expressed genes from transcriptome data with respect to their fold changes. FSEA identifies GO terms, which are shared by the group of genes with a similar magnitude of response, and assesses these changes. GO terms found by FSEA are fold-change-specifically (e.g. weakly, moderately or strongly) affected by a stimulus under investigation. We demonstrate that many responses to abiotic factors, mutations, treatments and diseases occur in a fold-change-specific manner. FSEA analyses suggest that there are two prevailing responses of functionally-related gene groups, either weak or strong. Notably, some of the fold-change-specific GO terms are invisible by classical algorithms for functional gene enrichment, SEA and GSEA. These are GO terms not enriched compared to the genome background but strictly regulated by a factor within specific fold-change intervals. FSEA analysis of a cancer-related transcriptome suggested that the gene groups with a tightly coordinated response can be the valuable source to search for possible regulators, markers and therapeutic targets in oncogenic processes. Availability and Implementation: FSEA is implemented as the FoldGO Bioconductor R package and a web-server https://webfsgor.sysbio.cytogen.ru/ .

Drought is one of the most serious abiotic stressors in the environment, restricting agricultural production by reducing plant growth, development, and productivity. To investigate such a complex and multifaceted stressor and its effects on the plants, a systems biology-based approach is necessitated, entailing the generation of co-expression networks, identification of highly-priority transcription factors (TFs) dynamic mathematical modeling, and computational simulations. Here, we studied a high-resolution drought transcriptome of Arabidopsis. We revealed distinct temporal transcriptional signatures and demonstrated the involvement of specific biological pathways. Generation of a large-scale co-expression network followed by network centrality analyses identified 117 TFs that possess critical properties of hubs, bottlenecks, and high clustering coefficients nodes. Dynamic transcriptional regulatory modeling on integrated TF-target and transcriptome datasets uncovered major transcriptional events during the course of drought stress. Mathematical transcriptional simulations allowed us to ascertain the activation status of major TFs as well as the transcriptional intensity and amplitude of their target genes. Finally, we validated our predictions by providing experimental evidence of gene expression under drought stress for a set of four TFs and their major target genes using qRT-PCR. Taken together, we provided a systems-level perspective on the dynamic transcriptional regulation during drought stress in Arabidopsis and uncovered numerous novel TFs that can potentially be used in future genetic crop engineering programs.

Cystic Fibrosis is a rare genetic disease that affects the transmission of chloride ions due to mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. Even though there are nearly 2000 mutations identified to be related to the condition, the most common mutation is F508del; deletion of a phenylalanine residue at 508. On the other hand, G542X which is a Class I mutation is also found very commonly and there are no modulator treatments available for it. Furthermore, it was investigated that R553X mutation can as well be corrected simultaneously with G542X mutation. Therefore, the main focus is on designing a gene therapy project that can correct all these three mutations at once by utilizing the prime editing technique via lipid-based delivery. In this way, the mutations can be edited through plasmids that were designed containing 2 pegRNAs and the Cas enzyme. To implement such an approach efficiently, both ex vivo, an animal model, and in vivo steps are to be designed. For the cell line, fibroblasts are selected due to their simplicity and low cost. The animal model of the experiment is determined to be a ferret concerning the high similarity to the human's CFTR protein and finally, the procedure will follow on a direct application in human Cystic Fibrosis patients. The plasmids are thought to be delivered through a cationic liposome that will reach the lungs with the aid of a nebulizer. At the last stage of the experimental procedure, Sanger Sequencing will be done to see if the desired edit within the CFTR has been performed successfully, and Next Generation Sequencing will be executed to see if there has been an off-target mutation in the remainder of the genome. Whereas for detecting the presence and expression of CFTR protein in humans, immunodetection with flow cytometry will be conducted.

Wheat gluten contains epitopes that trigger celiac disease (CD). A life-long strict gluten-free diet is the only treatment accepted for CD. However, very low-gluten wheat may provide an alternative treatment to CD. Conventional plant breeding methods are not sufficient to produce celiac-safe wheat. RNA interference technology, to some extent, succeeded in the development of safer wheat varieties. However, these varieties had multiple challenges in their implementation. Clustered Regularly Interspaced Short Palindromic Repeats-associated nuclease 9 (CRISPR/Cas9) is a versatile gene-editing tool that has the ability to edit the immunogenic gluten genes. So far, only a few studies have applied CRISPR/Cas9 to modify the wheat genome. In this article, we reviewed published literature that applied CRISPR/Cas9 in wheat genome editing to investigate the current status of the CRISPR/Cas9 system to produce a low-immunogenic wheat variety. We found that in recent years, the CRISPR/Cas9 system has been continuously improved to edit the complex hexaploid wheat genome. Although some reduced immunogenic wheat varieties have been reported, CRISPR/Cas9 has still not been fully explored in editing the wheat genome. We conclude that further studies are required to apply the CRISPR/Cas9 gene-editing system efficiently for the development of celiac-safe wheat variety and to establish it as a “tool to celiac safe wheat.”

A rather large portion of the population is sleep restricted in modern society, either by necessity or by choice. Epidemiological studies have observed that lack of sleep and poor sleep quality are associated with several pathological states, ranging from neuropsychiatric to metabolic diseases. To date, only a few studies have investigated the molecular consequences of sleep loss at the level of peripheral organs involved in maintaining metabolic homeostasis such as the white adipose tissue (WAT). From a molecular point of view, two key transcriptional regulators of WAT function are sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptors gamma (PPARγ). Both genes interact with specific targets involved in multiple metabolic processes, including adipocyte differentiation, browning and lipid metabolism. In this study, we assessed the effects of chronic sleep restriction (CSR) on the interplay between SIRT1 and PPARγ in mediating WAT metabolism in young adult mice. We found that CSR up-regulated expression of SIRT1 and some of its transcriptional targets involved in lipid metabolism and mitochondrial functions of adipocytes. Results suggest that CSR induces SIRT1-mediated metabolic remodeling of WAT to support an increased energy demand due to sleep loss.

We report the first correction by Prime editing of a mutation in the RYR1 gene, paving the way to gene therapies for RYR1-related myopathies. The RYR1 gene codes for a calcium channel named Ryanodine receptor 1, which is expressed in skeletal muscle fibers. The failure of this channel causes muscle weakness in patients, which degenerates into motor disabilities. Currently, there are no effective treatments for these diseases, which are mainly caused by point mutations. Prime editing allows the modification of precise nucleotides in the DNA. Our results showed a 59% correction rate of the T4709M mutation in the RYR1 gene in human myoblasts by RNA delivery of the Prime editing components. It is to be noted that the T4709M is recessive, and thus, persons having a heterozygous mutation are healthy. These results are the first demonstration that it is possible to correct mutations in the RYR1 gene.

Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA (tracrRNA), targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.

Improvement of agronomic traits in crops through gene editing (GE) relies on efficient trans-formation protocols for delivering the CRISPR/Cas9-coded transgenes. Recently, a few embryo-genesis-related genes have been described, the co-delivery of which significantly increases the transformation efficiency with reduced genotype dependency. Here we characterized the trans-genic and GE events in wheat (cv. Fielder) when transformed with the GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) chimeric gene. Trans-formation efficiency in our experiments ranged from 22% to 68%, and the editing events were faithfully propagated into the following generation. Both low and high copy number integration events were recovered in the T0 population with the various level of integrity of the left and right T-DNA borders. We also generated a population of wheat plants with ten different gRNAs targeting 30 loci in the genome. Comparison of the epigenetic profiles at the target sites and editing efficiency revealed a significant positive correlation between chromatin accessibility and muta-genesis rate. Overall, preliminary screening of the transgene quality and GE events in the T0 population of plants regenerated through the co-delivery of GRF-GIF can allow for the propagation of the best candidates for further phenotypic analysis.

The effect of Gymnema Sylvestre supplementation on beta cell and hepatic activity was explored in an alloxan-induced hyperglycemic rat model. Adult rats were made hyperglycemic via a single inj. (i.p) of Alloxan (120mg/kg b.w). Gymnema Sylvestre was supplemented @250mg/kg and 500mg/kg b.w. Blood glucose levels were constantly monitored. After 21 days, rats were euthanized, and blood and tissues (pancreas and liver) were collected for biochemical, expression, and histological analysis. One-way ANOVA was used to compare the means of different treatment groups. Gymnema Sylvestre significantly reduced blood glucose levels with a subsequent increase in plasma insulin levels in a dosage-dependent manner. Total oxidant status (TOS), malondialdehyde, LDL, VLDL, ALT, AST, triglyceride, total cholesterol, total protein, C-reactive protein, and cortisol levels were reduced significantly in alloxan-treated hyperglycemic rats supplemented with Gymnema Sylvestre as compared to control. Significantly raised paraoxonase, arylesterase, albumin, and HDL levels were also observed in Gymnema Sylvestre supplemented hyperglycemic rats. Increased mRNA expression of Ins-1, Ins-2, Gck, Pdx1, Mafa, and Pax6 were observed, while decreased expression of Cat, Sod1, Nrf2, and NF-kB was observed in the pancreas. Whereas increased mRNA expression of Gck, Irs1, SREBP1c, and Foxk1 and decreased expression of Irs2, ChREBP, Foxo1, and FoxA2 were observed in the liver. The current study indicates the potent effect of Gymnema Sylvestre on the transcription modulation of the insulin gene in the alloxan-induced hyperglycemic rat model. Enhanced plasma insulin levels further help to improve hyperglycemia-induced dyslipidemia through transcriptional modulation of hepatocytes.

Delta 9 tetrahydrocannabinol (Δ-9-THC or THC), the primary psychoactive constituent of marijuana, has been found to affect the human body in a variety of ways. According to the Substance Abuse and Mental Health Services Administration (SAMHSA) of the U.S. Department of Health and Human Services, the amount of THC in marijuana has steadily climbed over the past few decades, with today's marijuana having three times the concentration of THC compared to 25 years ago. Inhalation is a major route of exposure, allowing substances to enter the body via the respiratory tract. Therefore, it is imperative to understand the molecular underpinning of THC exposure in the lung epithelium, as lung cells receive significant amounts of THC during marijuana smoking. In this study, the transcriptional alterations caused by various concentrations of THC in non-cancerous human bronchial epithelial cell line (BEAS-2B) were studied. THC exposure (0, 800, 1000, 1200, and 1500 ng/mL) caused a significant dose-dependent decrease in cell viability after 24-hour exposure. Transcriptome analysis showed a distinct dose-dependent response. HIF-1 signaling, ferroptosis, AMPK signaling, and immunogenic pathways were activated by upregulated genes associated with dosing concentration of THC. Glutathione and fatty acid metabolic pathways were significantly altered by THC-dependent downregulated genes. and downregulated genes and their associated pathways in BEAS-2B cells. Ingenuity Pathway Analysis revealed several top canonical pathways altered by THC exposure, among which ferroptosis, NRF-2 mediated oxidative stress response, caveolar mediated endocytosis (loss of cell adhesion to the substrate), tumor microenvironment, HIF1alpha signaling, and the unfolded protein response pathways were the major pathways affected. The top genes that were significantly altered as a result of THC exposure were HMOX1, CDK7, HLA-C, and SLC39A4. Present results suggested that THC exposure can induce a variety of effects, including cell death, which may involve ferroptosis and the NRF-2 signaling pathway. Moreover, the Δ-9-THC-induced cell death was ameliorated by inhibiting the ferroptosis pathway. In contrast, the ferroptosis agonist exacerbated the cell death process, suggesting that Δ-9-THC utilizes the ferroptosis pathway to induce cell death in bronchial epithelial cells.

Pv11, an insect cell line established from the midge Polypedilum vanderplanki, is capable of ametabolic desiccation tolerance, so-called anhydrobiosis. We previously discovered that heat shock factor 1 (HSF1) contributes to the acquisition of desiccation tolerance by Pv11 cells, but the mechanistic details have yet to be elucidated. Here, by analyzing the gene expression profiles of newly established HSF1-knockout and -rescue cell lines, we show that HSF1 has a genome-wide effect on gene regulation in Pv11. HSF1-knockout cells exhibit a reduced desiccation survival rate, but this is completely restored in HSF1-rescue cells. By comparing mRNA profiles of the two cell lines, we reveal that HSF1 induces anhydrobiosis-related genes, especially genes encoding late embryogenesis abundant proteins and thioredoxins, but represses a group of genes involved in basal cellular processes, thus promoting an ametabolic state in the cell. In addition, HSF1 binding motifs are enriched in the promoters of anhydrobiosis-related genes and we demonstrate binding of HSF1 to these promoters by ChIP-qPCR. Thus, HSF1 directly regulates the transcription of anhydrobiosis-related genes and consequently plays a pivotal role in the induction of anhydrobiotic ability in Pv11 cells.

Fanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA patients display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore better therapies as well as new disease models are urgently needed. We have used CRISPR/Cas9 editing tools in order to interrupt the human FANCA gene by the generation of insertions/deletions (indels) in exon 4 in two cancer cell lines from sporadic HNSCC having no mutation in FA-genes: CAL27 and CAL33 cells. Our approach allowed efficient editing, subsequent purification of single-cell clones, and Sanger sequencing validation at the edited locus. Clones having frameshift indels in homozygosis did not express FANCA protein and were selected for further analysis. When compared with parental CAL27 and CAL33, FANCA-mutant cell clones displayed a FA-phenotype as they i) are highly sensitive to DNA interstrand crosslink (ICL) agents such as mitomycin C (MMC) or cisplatin, ii) do not monoubiquitinate FANCD2 upon MMC treatment and therefore iii) do not form FANCD2 nuclear foci, and iv) they display increased chromosome fragility and G2 arrest after diepoxybutane (DEB) treatment. These FANCA-mutant clones display similar growth rates as their parental cells. Interestingly, mutant cells acquire phenotypes associated with more aggressive disease, such as increased migration in wound healing assays. Therefore, CAL27 and CAL33 cells with FANCA mutations are phenocopies of FA-HNSCC cells.

The CCAAT/enhancer-binding protein β (C/EBPβ) is a transcription factor that regulates cellular proliferation, differentiation, apoptosis and tumorigenesis. Although the pro-oncogenic roles of C/EBPβ have been implicated in various human cancers, how it contributes to tumorigenesis or tumor progression has not been determined. Immunohistochemistry with human non-small cell lung cancer (NSCLC) tissues revealed that higher levels of C/EBPβ protein are expressed compared to normal lung tissues. Knockdown of C/EBPβ by siRNA reduced the proliferative capacity of NSCLC cells by delaying G₂/M transition of the cell cycle. In C/EBPβ-knockdown cells, a prolonged increase in phosphorylation of cyclin dependent kinase 1 at tyrosine 15 (Y15-pCDK1) was displayed with increased Wee1 and decreased Cdc25B expression, simultaneously. ChIP analysis showed that C/EBPβ bound to distal promoter regions of WEE1 and repressed WEE1 transcription through the interaction with histone deacetylase 2. Treatment of C/EBPβ-knockdown cells with a Wee1 inhibitor induced a decrease in Y15-pCDK1 and recovered cells from G₂/M arrest. In the xenograft tumors, the depletion of C/EBPβ significantly reduced tumor growth. Taken together, these results indicate that Wee1 is a novel transcription target of C/EBPβ that is required for the G₂/M phase of cell cycle progression, ultimately regulating proliferation of NSCLC cells.

The major auxin, indole-3-acetic acid (IAA), is associated with a plethora of growth and developmental processes including embryo development, expansion growth, cambial activity, and the induction of lateral root growth. Accumulation of the auxin precursor indole-3-acetamide (IAM) induces stress related processes by stimulating abscisic acid (ABA) biosynthesis. How IAM signaling is controlled is, at present, unclear. Here, we characterize an ami1/rooty double mutant, that we initially generated to study the metabolic and phenotypic consequences of a genetic blockade of the indole glucosinolate and IAM pathways in Arabidopsis thaliana. Our mass spectrometric analyses of the mutant revealed that the combination of the two mutations is not sufficient to fully prevent the conversion of IAM to IAA. The detected strong accumulation of IAM was, however, recognized to substantially impair seed development. We further show by genome-wide expression studies that the double mutant is broadly affected in its translational capacity, and that a small number of cell proliferation and plant growth regulating transcriptional circuits are repressed by the high IAM content in the seed. In accordance with the previously described growth reduction in response to elevated IAM levels, our data support the hypothesis that IAM is a growth repressing counterpart to IAA.

The regulatory mechanism of PCSK7 gene is still unknown, although its encoded protein PC7 is the most ancient and highly conserved of all proprotein convertases and exhibits enzymatic and non-enzymatic functions in liver triglyceride regulation. Bioinformatics algorithms were used to predict regulatory microRNAs (miRNAs) of PCSK7 expression. This led to the identification of 4 miRNAs, namely miR-125a-5p, miR-143-3p, miR-409-3p, and miR-320a-3p, with potential binding sites on the 3’-untranslated region (3′-UTR) of human PCSK7 mRNA. The expression patterns of these miRNAs and PCSK7 mRNA were assessed in three different cell lines with quantitative polymerase chain reaction (qPCR), which revealed reciprocal expression patterns between the expression levels of the four selected miRNAs and PCSK7. Next, the interactions and effects of these miRNAs on PCSK7 expression levels were investigated via cell-based expression analysis, dual-luciferase assay, and Western blot analysis. The data revealed that PCSK7 mRNA levels decreased in cells transfected with vectors overexpressing miR-125a-5p, miR-143-3p, and miR-409-3p, but not miR-320a-3p. The dual-luciferase assay demonstrated that the above 3 miRNAs could directly interact with putative target sites in PCSK7 3′-UTR and regulate its expression, whereas miR-320-3p exhibited no interaction. Western blot analysis further revealed that overexpression of miR-125a-5p in Huh7 cells inhibits the expression and ability of PC7 to cleave human transferrin receptor 1. Our results support a regulatory role of these miRNAs in PCSK7 expression and function and opens the way to assess their roles in the regulation of PC7 activity in vivo in the development of hepatic steatosis.