Gene Editing Technology Research Articles

Chromosomal instability (CIN) in HAP1 cell lines revealed by multiplex fluorescence in situ hybridisation (M-FISH)

BackgroundHAP1, a near-haploid human leukemic cancer cell line is often used in combination with CRISPR-Cas9 gene editing technology for genetic screens. HAP1 carries the Philadelphia chromosome (Ph) and an additional ~ 30 Mb fragment of chromosome 15 inserted into chromosome 19. The potential use of an in vitro cell line as a model system in biomedical research studies depends on its ability to maintain genome stability. Being a cancer cell line with a near-haploid genome, HAP1 is prone to genetic instability, which is further compounded by its tendency to diploidise in culture spontaneously. Moreover, CRISPR-Cas9 gene editing coupled with prolonged in-vitro cell culturing has the potential to induce unintended ‘off-target’ cytogenetic mutations.To gain an insight into chromosomal instability (CIN) and karyotype heterogeneity, 19 HAP1 cell lines were cytogenetically characterised, 17 of which were near-haploids and two double-haploids, using multiplex fluorescence in situ hybridisation (M-FISH), at single cell resolution. We focused on novel numerical (N) and structural (S) CIN and discussed the potential causal factors for the observed instability. For each cell line we examined its ploidy, gene editing status and its length of in-vitro cell culturing.ResultsSixteen of the 19 cell lines had been gene edited with passage numbers ranging from 10 to 35. Diploidisation in 17 near-haploid cell lines ranged from 4 to 35% and percentage of N- and S-CIN in [1n] and [2n] metaphases ranged from 7 to 50% with two cell lines showing no CIN. Percentage of cells with CIN in the two double-haploid cell lines were 96% and 100% respectively. The most common S-CIN observed was deletion followed by translocation of both types, non-reciprocal and Robertsonian. Interestingly, we observed a prevalence of S-CIN associated with chromosome 13 in both near-and double-haploid cell lines, with a high incidence of Robertsonian translocation involving chromosome 13. Furthermore, locus-specific BAC (bacterial artificial chromosome) FISH enabled us to show for the first time that the additional chromosome 15 fragment is inserted into the p-arm rather than the q-arm of chromosome 19 of the HAP1 genome.ConclusionOur study revealed a high incidence of CIN leading to karyotype heterogeneity in majority of the HAP1 cell lines with the number of chromosomal aberrations varying between cell lines. A noteworthy observation was the high frequency of structural chromosomal aberrations associated with chromosome 13. We showed that CRISPR-Cas9 gene editing technology in combination with spontaneous diploidisation and prolonged in-vitro cell culturing is potentially instrumental in inducing further chromosomal rearrangements in the HAP1 cell lines with existing CIN. We highlight the importance of maintaining cell lines at low passage and the need for regular monitoring to prevent implications in downstream applications. Our study also established that the additional fragment of chromosome 15 in the HAP1 genome is inserted into chromosome 19p rather than 19q.

With great power comes great responsibility: why ‘safe enough’ is not good enough in debates on new gene technologies

New genomic techniques (NGTs) are powerful technologies with the potential to change how we relate to our food, food producers, and natural environment. Their use may affect the practices and values our societies are built on. Like many countries, the EU is currently revisiting its GMO legislation to accommodate the emergence of NGTs. We argue that assessing such technologies according to whether they are ‘safe enough’ will not create the public trust necessary for societal acceptance. To avoid past mistakes of under- or miscommunication about possible impacts, we need open, transparent, and inclusive societal debate on the nature of the science of gene (editing) technologies, on how to use them, and whether they contribute to sustainable solutions to societal and environmental challenges. To be trustworthy, GMO regulation must demonstrate the authorities’ ability to manage the scientific, socio-economic, environmental, and ethical complexities and uncertainties associated with NGTs. Regulators and authorities should give equal attention to the reflexive and the emotional aspects of trust and make room for honest public and stakeholder inclusion processes. The European Group of Ethics in Science and Technology’s recent report on the Ethics of Genome Editing (2021) is important in calling attention to a series of fundamental issues that ought to be included in debates on the regulation and use of NGTs to ensure public trust in these technologies and in regulating authorities. With the great power of NGTs comes great responsibility, and the way forward must be grounded in responsible research, innovation, and regulation.

Uncovering the Genetic of Cadmium Accumulation in the Rice 3K Panel.

Because Cadmium (Cd) is harmful to humans, and most non-smokers are exposed to Cd mainly through rice consumption, low-Cd rice breeding is urgently needed. It might not be possible to apply variation created using gene editing technology to breeding directly, so it is important to explore genetic variation in this trait in a natural population. In this study, variation in 4 genes was identified among 3024 accessions from the International Rice Research Institute 3000 Rice Genome Project (IRRI 3K-RGP) and 71 other important varieties, and the relationships between the variants and plant Cd accumulation were validated with hydroponic and pot experiments. Variants in OsNRAMP1, OsNRAMP5, OsLCD, and OsHMA3 were grouped into two, four, three, and two haplotypes, respectively. Fourteen combinations of these haplotypes, which were referred to as Cd-mobile types, were found in the collection. Of these, type 14 was shown to have the greatest potential for low-Cd accumulation, and functional markers for this type were designed. The results of this study provide an important resource for low-Cd rice breeding and highlight an effective strategy for pre-breeding programs.

Activation of the cGAS-STING pathway combined with CRISPR-Cas9 gene editing triggering long-term immunotherapy

Effective activation of cGAS-STING pathway combined with immune checkpoint blockade (ICB) within the immunosuppressive tumor microenvironment to induce stronger immune responsiveness yet remains challenging. CRISPR-Cas9 gene editing technology, which offers the benefits of permanence and irreversibility, could recognize the target genome sequence with sgRNA (Guide RNA) and guide the Cas9 protease to knock down the target gene. Herein, a nanoplatform (HMnMPH) for dual activation of cGAS-STING pathway in combination with CRISPR-Cas9 gene editing to silence programmed death ligand 1 (PD-L1) to trigger long-term immunotherapy was reported. The HMnMPH consists of hollow manganese dioxide (HMn) loaded with STING agonist (MSA-2) and CRISPR-Cas9/sg-PD-L1 plasmid with further modification of hyaluronic acid (HA). In acidic and GSH overexpressed tumor environment, HMnPMH was degraded to release large amounts of Mn ions and STING agonists, strongly and persistently activating the cGAS-STING pathway to promote the release of type I interferon and pro-inflammatory factors. Meanwhile, the released CRISPR-Cas9 plasmid could knockdown the PD-L1 immune checkpoint and restart immunosuppressive T cells to differentiate into cytotoxic T lymphocytes significantly, which reduced the activity of primary and distal tumors and demonstrated a long-term immune memory effect on distal tumors.

Gene regulatory and gene editing tools and their applications for retinal diseases and neuroprotection: From proof-of-concept to clinical trial.

Gene editing and gene regulatory fields are continuously developing new and safer tools that move beyond the initial CRISPR/Cas9 technology. As more advanced applications are emerging, it becomes crucial to understand and establish more complex gene regulatory and editing tools for efficient gene therapy applications. Ophthalmology is one of the leading fields in gene therapy applications with more than 90 clinical trials and numerous proof-of-concept studies. The majority of clinical trials are gene replacement therapies that are ideal for monogenic diseases. Despite Luxturna's clinical success, there are still several limitations to gene replacement therapies including the size of the target gene, the choice of the promoter as well as the pathogenic alleles. Therefore, further attempts to employ novel gene regulatory and gene editing applications are crucial to targeting retinal diseases that have not been possible with the existing approaches. CRISPR-Cas9 technology opened up the door for corrective gene therapies with its gene editing properties. Advancements in CRISPR-Cas9-associated tools including base modifiers and prime editing already improved the efficiency and safety profile of base editing approaches. While base editing is a highly promising effort, gene regulatory approaches that do not interfere with genomic changes are also becoming available as safer alternatives. Antisense oligonucleotides are one of the most commonly used approaches for correcting splicing defects or eliminating mutant mRNA. More complex gene regulatory methodologies like artificial transcription factors are also another developing field that allows targeting haploinsufficiency conditions, functionally equivalent genes, and multiplex gene regulation. In this review, we summarized the novel gene editing and gene regulatory technologies and highlighted recent translational progress, potential applications, and limitations with a focus on retinal diseases.

Plant Resistance to Fungal Pathogens: Bibliometric Analysis and Visualization.

Plants are susceptible to fungal pathogen infection, threatening plant growth and development. Researchers worldwide have conducted extensive studies to address this issue and have published numerous articles on the subject, but they lack a scientometric evaluation. This study analyzed international research on the topic “Plant resistance to fungal pathogens” between 2008 and 2021, using the core database of the Web of Science (WoS). By searching the subject words “Plants”, “Disease Resistance”, and “Fungal Pathogens”, we received 6687 articles. Bibliometric visualization software analyzes the most published countries, institutions, journals, authors, the most cited articles, and the most common keywords. The results show that the number of articles in the database has increased year by year, with the United States and China occupying the core positions, accounting for 46.16% of the total published articles worldwide. The United States Department of Agriculture (USDA) is the main publishing organization. Wang Guoliang is the author with the most published articles, and the Frontiers in Plant Science ranks first in published articles. The research on plant anti-fungal pathogens is booming, and international exchanges and cooperation need to be further strengthened. This paper summarizes five possible research ideas, from fungal pathogens, gene editing technology, extraction of secondary metabolites from plants as anti-fungal agents, identification of related signal pathways, fungal molecular databases, and development of nanomaterials, to provide data for related research.

Detection of Tropical Diseases Caused by Mosquitoes Using CRISPR-Based Biosensors.

Tropical diseases (TDs) are among the leading cause of mortality and fatality globally. The emergence and reemergence of TDs continue to challenge healthcare system. Several tropical diseases such as yellow fever, tuberculosis, cholera, Ebola, HIV, rotavirus, dengue, and malaria outbreaks have led to endemics and epidemics around the world, resulting in millions of deaths. The increase in climate change, migration and urbanization, overcrowding, and other factors continue to increase the spread of TDs. More cases of TDs are recorded as a result of substandard health care systems and lack of access to clean water and food. Early diagnosis of these diseases is crucial for treatment and control. Despite the advancement and development of numerous diagnosis assays, the healthcare system is still hindered by many challenges which include low sensitivity, specificity, the need of trained pathologists, the use of chemicals and a lack of point of care (POC) diagnostic. In order to address these issues, scientists have adopted the use of CRISPR/Cas systems which are gene editing technologies that mimic bacterial immune pathways. Recent advances in CRISPR-based biotechnology have significantly expanded the development of biomolecular sensors for diagnosing diseases and understanding cellular signaling pathways. The CRISPR/Cas strategy plays an excellent role in the field of biosensors. The latest developments are evolving with the specific use of CRISPR, which aims for a fast and accurate sensor system. Thus, the aim of this review is to provide concise knowledge on TDs associated with mosquitoes in terms of pathology and epidemiology as well as background knowledge on CRISPR in prokaryotes and eukaryotes. Moreover, the study overviews the application of the CRISPR/Cas system for detection of TDs associated with mosquitoes.

Intracranial Delivery of Lipid Nanoparticles in the Murine Model

INTRODUCTION: Lipid nanoparticles (LNPs) offer several benefits over viral vectors for delivery of gene editing technology, including lower antigenicity and higher biocompatibility, scalability, modifiability, and the ability to package more variable cargo. In addition, their lipophilic nature allows them to target difficult-to-transduce cell populations, making them ideal for brain delivery. In this study, we demonstrate that intracranial (IC) delivery of LNPs is an effective method for brain targeting. METHODS:ROSA26mtmg mice at postnatal day 0 were injected with LNPs containing Cre-recombinase mRNA (LNP-Cre) or saline directly into the intraventricular space; these mice express tdTomato (red) fluorescence at baseline, which is excised in the presence of Cre-recombinase to express eGFP (green) fluorescence. Mice received 100 ng of LNP-Cre (n = 4) or saline (n = 1) in 4 μL total volume bilaterally. The pups were monitored for 10 days and then euthanized for analysis. RESULTS: At harvest, all pups were viable with no gross neurologic abnormalities. Brains injected with LNP-Cre showed strong eGFP positivity on stereomicroscopy (Fig. 1A) compared with control (Fig. 1B) and eGFP+ cells lining the ventricular spaces on histology (Fig. 1C). Quantitative polymerase chain reaction showed an average 20-fold increase in eGFP mRNA expression in the cortex, 16-fold increase in the midbrain, and 3-fold increase in the cerebellum compared with control (Fig. 1D).Figure 1CONCLUSION: This study shows that IC LNP injection is a safe and effective technique for brain editing with 100% survival over 10 days. Further optimization is critical for use in preclinical gene editing studies for neurocognitive disorders where brain targeting is the primary focus.

HPGDS is a novel prognostic marker associated with lipid metabolism and aggressiveness in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most common respiratory globallywith a poor prognosis. Lipid metabolism is extremely important for the occurrence and development of cancer. However, the role of genes involved in lipid metabolism in LUAD development is unclear. We aimed to identify the abnormal lipid metabolism pathway of LUAD, construct a novel prognostic model of LUAD, and discover novel biomarkers involved in lipid metabolism inLUAD. Based on differentially expressed genes involved in lipid metabolism in LUAD samples from The Cancer Genome Atlas (TCGA), abnormal lipid metabolism pathways in LUAD were analyzed. The lasso penalized regression analysis was performed on the TCGA cohort (training set) to construct a risk score formula. The predictive ability of the risk score was validated in the Gene Expression Omnibus (GEO) dataset (validation set) using Kaplan-Meier analysis and ROC curves. Finally, based on CRISPR gene editing technology, hematopoietic prostaglandin D synthase (HPGDS) was knocked out in A549 cell lines, the changes in lipid metabolism-related markers were detected by western blotting, and the changes in cell migration were detected by transwellassay. Based on the differential genes between lung cancer tissue and normal tissue, we found that the arachidonic acid metabolism pathway is an abnormal lipid metabolism pathway in both lung adenocarcinoma and lung squamous cell carcinoma. Based on the sample information of TCGA and abnormally expressed lipid metabolism-related genes, a 9-gene prognostic risk score was successfully constructed and validated in the GEO dataset. Finally, we found that knockdown of HPGDS in A549 cell lines promoted lipid synthesis and is more invasive than in control cells. Rescue assays showed that ACSL1 knockdown reversed the pro-migration effects of HPGDS knockdown. The knockdown of HPGDS promoted migration response by upregulating the expression of the lipid metabolism key enzymes ACSL1 and ACC. The genes involved in lipid metabolism are associated with the occurrence and development of LUAD. HPGDS can be a therapeutic target of a potential lipid metabolism pathway in LUAD, and the therapeutic target of lipid metabolism genes in LUAD should be studied further.

Gene Editing in Human Haematopoietic Stem Cells for the Treatment of Primary Immunodeficiencies.

In recent years, gene-editing technologies have revolutionised precision medicine, and human trials of this technology have been reported in cell-based cancer therapies and other genetic disorders. The same techniques have the potential to reverse mutations in monogenic primary immunodeficiencies (PIDs), and transplantation of edited haematopoietic stem cells may provide a functional cure for these diseases. In this review, we discuss the methods of gene editing being explored and describe progress made so far with several PIDs. We also detail the remaining challenges, how to confidently detect off-target effects and chromosomal abnormalities in a timely manner, how to obtain long-term benefits, and how to achieve physiological levels of expression of the therapeutic gene. With advances in gene editing, we envisage a robust clinical translation of this technology in the coming decade.

Application of advanced technology in traditional Chinese medicine for cancer therapy.

Although cancer has seriously threatened people’s health, it is also identified by the World Health Organization as a controllable, treatable and even curable chronic disease. Traditional Chinese medicine (TCM) has been extensively used to treat cancer due to its multiple targets, minimum side effects and potent therapeutic effects, and thus plays an important role in all stages of tumor therapy. With the continuous progress in cancer treatment, the overall efficacy of cancer therapy has been significantly improved, and the survival time of patients has been dramatically prolonged. In recent years, a series of advanced technologies, including nanotechnology, gene editing technology, real-time cell-based assay (RTCA) technology, and flow cytometry analysis technology, have been developed and applied to study TCM for cancer therapy, which efficiently improve the medicinal value of TCM and accelerate the research progress of TCM in cancer therapy. Therefore, the applications of these advanced technologies in TCM for cancer therapy are summarized in this review. We hope this review will provide a good guidance for TCM in cancer therapy.

Gene editing of Duchenne muscular dystrophy using biomineralization-based spCas9 variant nanoparticles

The CRISPR/Cas9 mediated genome editing have provided a promising strategy to correct multiple mutations of Duchenne muscular dystrophy (DMD). However, the delivery of CRISPR/Cas9 system into mammalian cell for DMD gene editing mainly relies on adeno associated virus (AAV)-mediated transport. Meanwhile, the protospacer adjacent motif (PAM) requirement of wild-typed Cas9 protein causing the target sites for exon splice acceptor site are restricted to limited regions. Here, we developed a biomineralized PAMLess Cas9 (SpRY) variant nanoparticles (Bm-SpRY NPs) for DMD gene editing in vitro and in vivo. This method described a facile synthesis of biomineralized NPs with high SpRY pDNA encapsulation efficiency. In vitro results show that the Bm-SpRY NPs have the obvious advantages of well biocompatibility and protecting SpRY pDNA from enzyme degradation and efficient delivery under high serum condition. Cell studies demonstrated that Bm-SpRY NPs enable rapid cellular uptake, endo-lysosomes escape and nucleus transport. Meanwhiles, the DMD gene editing via Bm-SpRY NPs pathway is transient process without genomic integration. We evaluated multiple target regions with different PAMs for the DMD exon 51 splice acceptor site through Bm-SpRY NPs method and found that the target region with TAG PAM has the highest editing efficiency and significant preferential mutation. In vivo results show that intramuscular injection of Bm-SpRY NPs enable DMD gene mutation in muscle tissue without tissue damage. This study may extend the advanced application of CRISPR system for DMD therapy. Statement of significanceThe gene editing technology of CRISPR/Cas9 provides an effective treatment strategy for the Duchenne muscular dystrophy (DMD) therapy. However, the delivery of CRISPR system in mammalian cell mainly relies on viral mediated transport and the NGG or NAG requirement of wild-typed Cas9 protein limits the target region in DMD gene. Here, the present study provides a biomineralized PAM Less Cas9 (SpRY) variant nanoparticles (Bm-SpRY NPs) for DMD gene editing in vitro and in vivo. This study may extend the application of CRISPR system for DMD gene therapy.

Coevolution of tandemly repeated hlips and RpaB-like transcriptional factor confers desiccation tolerance to subaerial Nostoc species

Desert-inhabiting cyanobacteria can tolerate extreme desiccation and quickly revive after rehydration. The regulatory mechanisms that enable their vegetative cells to resurrect upon rehydration are poorly understood. In this study, we identified a single gene family of high light-inducible proteins (Hlips) with dramatic expansion in the Nostoc flagelliforme genome and found an intriguingly special convergence formed through four tandem gene duplication. The emerged four independent hlip genes form a gene cluster (hlips-cluster) and respond to dehydration positively. The gene mutants in N. flagelliforme were successfully generated by using gene-editing technology. Phenotypic analysis showed that the desiccation tolerance of hlips-cluster-deleted mutant decreased significantly due to impaired photosystem II repair, whereas heterologous expression of hlips-cluster from N. flagelliforme enhanced desiccation tolerance in Nostoc sp. PCC 7120. Furthermore, a transcription factor Hrf1 (hlips-cluster repressor factor 1) was identified and shown to coordinately regulate the expression of hlips-cluster and desiccation-induced psbAs. Hrf1 acts as a negative regulator for the adaptation of N. flagelliforme to the harsh desert environment. Phylogenetic analysis revealed that most species in the Nostoc genus possess both tandemly repeated Hlips and Hrf1. Our results suggest convergent evolution of desiccation tolerance through the coevolution of tandem Hlips duplication and Hrf1 in subaerial Nostoc species, providing insights into the mechanism of desiccation tolerance in photosynthetic organisms.

In vivo correction of cystic fibrosis mediated by PNA nanoparticles.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We sought to correct the multiple organ dysfunction of the F508del CF-causing mutation using systemic delivery of peptide nucleic acid gene editing technology mediated by biocompatible polymeric nanoparticles. We confirmed phenotypic and genotypic modification in vitro in primary nasal epithelial cells from F508del mice grown at air-liquid interface and in vivo in F508del mice following intravenous delivery. In vivo treatment resulted in a partial gain of CFTR function in epithelia as measured by in situ potential differences and Ussing chamber assays and correction of CFTR in both airway and GI tissues with no off-target effects above background. Our studies demonstrate that systemic gene editing is possible, and more specifically that intravenous delivery of PNA NPs designed to correct CF-causing mutations is a viable option to ameliorate CF in multiple affected organs.

Recent advances in neuropeptide-related omics and gene editing: Spotlight on NPY and somatostatin and their roles in growth and food intake of fish.

Feeding and growth are two closely related and important physiological processes in living organisms. Studies in mammals have provided us with a series of characterizations of neuropeptides and their receptors as well as their roles in appetite control and growth. The central nervous system, especially the hypothalamus, plays an important role in the regulation of appetite. Based on their role in the regulation of feeding, neuropeptides can be classified as orexigenic peptide and anorexigenic peptide. To date, the regulation mechanism of neuropeptide on feeding and growth has been explored mainly from mammalian models, however, as a lower and diverse vertebrate, little is known in fish regarding the knowledge of regulatory roles of neuropeptides and their receptors. In recent years, the development of omics and gene editing technology has accelerated the speed and depth of research on neuropeptides and their receptors. These powerful techniques and tools allow a more precise and comprehensive perspective to explore the functional mechanisms of neuropeptides. This paper reviews the recent advance of omics and gene editing technologies in neuropeptides and receptors and their progresses in the regulation of feeding and growth of fish. The purpose of this review is to contribute to a comparative understanding of the functional mechanisms of neuropeptides in non-mammalians, especially fish.

CD47KO/CRT dual-bioengineered cell membrane-coated nanovaccine combined with anti-PD-L1 antibody for boosting tumor immunotherapy.

Tumor vaccines trigger tumor-specific immune responses to prevent or treat tumors by activating the hosts’ immune systems, and therefore, these vaccines have potential clinical applications. However, the low immunogenicity of the tumor antigen itself and the low efficiency of the vaccine delivery system hinder the efficacy of tumor vaccines that cannot produce high-efficiency and long-lasting antitumor immune effects. Here, we constructed a nanovaccine by integrating CD47KO/CRT dual-bioengineered B16F10 cancer cell membranes and the unmethylated cytosine-phosphate-guanine (CpG) adjuvant. Hyperbranched PEI25k was used to load unmethylated cytosine-phosphate-guanine (CpG) through electrostatic adsorption to prepare PEI25k/CpG nanoparticles (PEI25k/CpG-NPs). CD47KO/CRT dual-bioengineered cells were obtained by CRISPR-Cas9 gene editing technology, followed by the cell surface translocation of calreticulin (CRT) to induce immunogenic cell death (ICD) in vitro. Finally, the extracted cell membranes were coextruded with PEI25k/CpG-NPs to construct the CD47KO/CRT dual-bioengineered cancer cell membrane-coated nanoparticles (DBE@CCNPs). DBE@CCNPs could promote endocytosis of antigens and adjuvants in murine bone marrow derived dendritic cells (BMDCs) and induce their maturation and antigen cross-presentation. To avoid immune checkpoint molecule-induced T cell dysfunction, the immune checkpoint inhibitor, the anti-PD-L1 antibody, was introduced to boost tumor immunotherapy through a combination with the DBE@CCNPs nanovaccine. This combination therapy strategy can significantly alleviate tumor growth and may open up a potential strategy for clinical tumor immunotherapy.

Gene Therapy for Human Diseases: Recent Achievements and Near-Term Development Prospects

The article briefly summarizes recent advances in genetic medicine that paved the way for the further development of gene therapy and set the stage for the development of next generation technology. Issues related to the main obstacle for wider application of gene therapy methods, in particular, with the immune response to gene delivery vectors and transgene products are considered. In this context, the role of new technology allowing to bypass the immune obstacle, such as development of modified capsids of adeno-associated viruses (AAV) and methods for temporary removal of antibodies from the bloodstream, as well as gene transfer into tissues using nanoparticles, is discussed. Along with the technology of the first generation gene therapy focused on the delivery of transgenes into target tissues, latest advances in the development of a completely new approach to gene therapy which is based on precise modification of the human genome sequence, gene editing technology, are summarized. Finally, promising next-generation gene editing technology is outlined, such as RNA-targeted editing technology and epigenome editing technology, which are more specific, precise, efficient and applicable to different groups of diseases. The article concludes that gene therapy and, in particular, human genome editing is perhaps the most exciting and revolutionary biotechnology of our time, due to both recent developments and opportunities it might provide in the nearest future.

Generation of PCBP1-deficient pigs using CRISPR/Cas9-mediated gene editing

SummaryClassical swine fever virus (CSFV), a classic swine fever pathogen, causes severe economic losses worldwide. Poly (rC)-binding protein 1 (PCBP1), which interacts with Npro of CSFV, plays a vital role in CSFV growth. We are the first to report the generation of PCBP1-deficient pigs via gene-editing technology. The PCBP1-deficient pigs exhibited normal birth weight and reproductive-performance traits and developed normally. Viral challenge experiments indicated that primary cells isolated from F0- and F1-generation pigs exhibited significantly reduced CSFV infection. Additional mechanistic exploration further confirmed that the PCBP1 deficiency-mediated antiviral effect is related to the activation of type I interferon (IFN). Besides showing that a gene-editing strategy could be used to generate PCBP1-deficient pigs, our study introduces a valuable animal model for further investigating the infection mechanisms of CSFV that will help to develop better antiviral solutions.

How do consumers respond to labels for crispr (gene-editing)?

Although it is accepted that communication about biotechnology influences consumers' willingness to pay for food produced using genetic engineering technology, communication on subjects such as genetic technology is complex. With more ways to communicate with consumers following the rise of media, especially social media, providing such complex information is feasible. This paper delivers actionable policy recommendations based on the effect of different information delivery methods on consumers' preference for orange juice produced using different production methods. Our results suggest that consumers have less knowledge of CRISPR (gene-editing) technology than GM (genetic modification) technology. Communication through infographics and video led to larger changes in willingness to pay than through text descriptions (which are often used in survey research). In general, providing information about the types of biotechnology helps consumers differentiate between the two technologies. With more information, consumers are willing to pay more for CRISPR orange juice than GM orange juice. The results suggest institutions and policy-makers use infographic information to better communicate with consumers. Researchers should also consider the potential effects of communication methods for information treatments in choice experiments.

Non-small-molecule therapeutics for drug addiction: From pharmacokinetics modulating to synthetic biology

Drug addiction is a pervasive problem worldwide. It not only affects the abuser's life, but also poses a serious threat to public health. Accordingly, there is a strong demand for the development of novel and effective therapies for drug addiction. Considering that small-molecule drugs have only had limited success, there is great interest in developing alternative strategies that extend the reach of small-molecule-based therapies. The antibody-based trapping approach and the enzyme catalytic strategy have been considered as promising ways to reduce the euphoria of drug users by altering drug pharmacokinetics and decreasing drug concentrations in the central nervous system. However, these biological macromolecules are generally unstable and their in vivo half-lives are short. With the rapid development of gene editing technologies, it is possible to perform ex vivo gene therapy for the long-term and stable delivery of enzymes and other effector proteins, which could free abusers from frequent injections of therapeutics. By constructing programmed gene switches that regulate spatiotemporal gene expression in response to illicit drugs, this perspective proposed the concept of preventing drug addiction by the drug itself. This strategy enables the controlled release of therapeutic proteins in vivo and is expected to improve patient safety and compliance. This will open up new opportunities for next-generation medicine and hold great promise for expanding our ability to treat drug addiction.