Clustered regularly interspaced short palindromic repeat sequences(CRISPR)and their accompanying proteins(Cas),commonly presenting in bacteria and archaea,make up the CRISPR/Cas system.As one of the funda-mental sourc...Clustered regularly interspaced short palindromic repeat sequences(CRISPR)and their accompanying proteins(Cas),commonly presenting in bacteria and archaea,make up the CRISPR/Cas system.As one of the funda-mental sources of nutrition for humans,edible crops play a crucial role in ensuring global food security.CRISPR/Cas9 gene editing has been applied to improve many crop traits,such as increasing nitrogen utilization efficiency,creating male sterile germplasm,and regulating tiller and spikelet formation.This paper provides a comprehensive overview of the use of CRISPR/Cas gene editing technology in crop genomes,covering the targeted genes,the types of editing that take place,the mechanism of action.Finally,we also discussed the efficiency of gene editing and pointed the future direction on how to speed up crop molecular breeding,increase breeding effectiveness,and produce more new crop varieties with high qualities.展开更多
The potential of regenerative medicine in the clinical space is vast,given its ability to repair and replace damaged tissues,restore lost functions due to age or disease,and transform personalized therapy.Traditional ...The potential of regenerative medicine in the clinical space is vast,given its ability to repair and replace damaged tissues,restore lost functions due to age or disease,and transform personalized therapy.Traditional regenerative medicine and tissue engineering strategies have created specialized tissues using progenitor cells and various biological stimuli.To date,there are many US Food and Drug Administration(FDA)-approved regenerative medicine therapies,such as those for wound healing and orthopedic injuries.Nonetheless,these therapies face challenges,including off-target effects,a lack of precision,and failure to target the disease or injury at its origin.In search of novel,precise,and efficient alternatives,the regenerative medicine landscape is shifting towards genome engineering technologies,particularly gene editing.Clustered regularly interspaced short palindromic repeats(CRISPR)-based gene editing systems enable precise knock-ins,knockouts,transcriptional activation and repression,as well as specific base conversions.This advancement has allowed researchers to treat genetic and degenerative diseases,control cell fate for highly regulated tissue repair,and enhance tissue functions.In this review,we explore the progress and future prospects of CRISPR technologies in regenerative medicine,focusing on how gene editing has led to advanced therapeutic applications and served as a versatile research tool for understanding tissue development and disease progression.展开更多
Nonobstructive azoospermia(NOA)affects about 60%of men with azoospermia,representing a severe form of male infertility.The current approach to manage NOA primarily involves testicular sperm retrieval methods such as c...Nonobstructive azoospermia(NOA)affects about 60%of men with azoospermia,representing a severe form of male infertility.The current approach to manage NOA primarily involves testicular sperm retrieval methods such as conventional testicular sperm extraction(c-TESE)and microdissection testicular sperm extraction(micro-TESE).While combining testicular sperm retrieval with intracytoplasmic sperm injection(ICSI)offers hope for patients,the overall sperm retrieval rate(SRR)stands at around 50%.In cases where micro-TESE fails to retrieve sperm,limited options,like donor sperm or adoption,can be problematic in certain cultural contexts.This paper delves into prospective treatments for NOA management.Gene editing technologies,particularly clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)protein 9(CRISPR/Cas9),hold potential for correcting genetic mutations underlying testicular dysfunction.However,these technologies face challenges due to their complexity,potential off-target effects,ethical concerns,and affordability.This calls for research to address key challenges associated with NOA management within the clinical settings.This also necessitate ongoing research essential for developing more sensitive diagnostic tests,validating novel treatments,and customizing current treatment strategies for individual patients.This review concluded that the future of NOA management may entail a combination of these treatment options,tailored to each patient’s unique circumstances,providing a comprehensive approach to address NOA challenges.展开更多
Generation of mutants with clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA(mRN...Generation of mutants with clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA(mRNA)or protein and transcribed guide RNA(gRNA).However,the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present.In this study,we employed a poly-transfer RNA(tRNA)-gRNA(PTG)system driven by cytomegalovirus(CMV)promoter to target the medaka(Oryzias latipes)endogenous gene tyrosinase(tyr)or paired box 6.1(pax6.1)and illustrated its function in a medaka cell line and embryos.The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka.This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.展开更多
Generation of mouse models carrying a defined point mutation,especially disease-related point mutations,is of considerable interest for research in biology and medicine.The standard method based on embryonic stem cell...Generation of mouse models carrying a defined point mutation,especially disease-related point mutations,is of considerable interest for research in biology and medicine.The standard method based on embryonic stem cell（ESC）-mediated homologous recombination（HR）is time-and labor-consuming.展开更多
Metabolic engineering of the tumor microenvironment has emerged as a new strategy.Lactate dehydrogenase A(LDHA)is a prominent target for metabolic engineering.Here,we designed a cationic lipid nanoparticle formulation...Metabolic engineering of the tumor microenvironment has emerged as a new strategy.Lactate dehydrogenase A(LDHA)is a prominent target for metabolic engineering.Here,we designed a cationic lipid nanoparticle formulation for LDHA gene editing.The plasmid DNA delivery efficiency of our lipid nanoparticle formulations was screened by testing the fluorescence of lipid nanoparticles complexed to plasmid DNA encoding green fluorescence protein(GFP).The delivery efficiency was affected by the ratios of three components:a cationic lipid,cholesterol or its derivative,and a fusogenic lipid.The lipid nanoparticle designated formulation F3 was complexed to plasmid DNA co-encoding CRISPR-associated protein 9 and LDHA-specific sgRNA,yielding the lipoplex,pCas9-sgLDHA/F3.The lipoplex including GFP-encoding plasmid DNA provided gene editing in HeLa-GFP cells.Treatment of B16F10 tumor cells with pCas9-sgLDHA/F3 yielded editing of the LDHA gene and increased the pH of the culture medium.pCas9-sgLDHA/F3 treatment activated the interferon-gamma and granzyme production of T cells in culture.In vivo,combining pCas9-sgLDHA/F3 with immune checkpoint-inhibiting anti-PD-L1 antibody provided a synergistic antitumor effect and prolonged the survival of tumor model mice.This study suggests that combining metabolic engineering of the tumor microenvironment with immune checkpoint inhibition could be a valuable antitumor strategy.展开更多
Although tremendous efforts have been made to prevent and treat HIV-1 infection,HIV-1/AIDS remains a major threat to global human health.The combination antiretroviral therapy(cART),although able to suppress HIV-1 rep...Although tremendous efforts have been made to prevent and treat HIV-1 infection,HIV-1/AIDS remains a major threat to global human health.The combination antiretroviral therapy(cART),although able to suppress HIV-1 replication,cannot eliminate the proviral DNA integrated into the human genome and thus requires lifelong treatment that may lead to various side effects.In recent years,clustered regularly interspaced short palindromic repeat(CRISPR)-associated nuclease 9(Cas9)related gene-editing systems have been developed and designed as effective ways to treat HIV-1 infection.However,new gene-targeting tools derived from or functioning like CRISPR/Cas9,including base editor,prime editing,SHERLOCK,DETECTR,PAC-MAN,ABACAS,pfAGO,have been developed and optimized for pathogens detection and diseases correction.Here,we summarize recent studies on HIV-1/AIDS gene therapy and provide more gene-editing targets based on studies relating to the molecular mechanism of HIV-1 infection.We also identify the strategies and potential applications of these new gene-editing technologies for HIV-1/AIDS treatment in the future.Moreover,we discuss the caveats and problems that should be addressed before the clinical use of these versatile CRISPR-based gene targeting tools.Finally,we offer alternative solutions to improve the practice of gene targeting in HIV-1/AIDS gene therapy.展开更多
Plant breeding is well recognized as one of the most important means to meet food security challenges caused by the ever-increasing world population. During the past three decades, plant breeding has been empowered by...Plant breeding is well recognized as one of the most important means to meet food security challenges caused by the ever-increasing world population. During the past three decades, plant breeding has been empowered by both new knowledge on trait development and regulation(e.g., functional genomics) and new technologies(e.g., biotechnologies and phenomics). Gene editing, particularly by clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas) and its variants, has become a powerful technology in plant research and may become a game-changer in plant breeding. Traits are conferred by coding and non-coding genes. From this perspective, we propose different editing strategies for these two types of genes. The activity of an encoded enzyme and its quantity are regulated at transcriptional and post-transcriptional, as well as translational and post-translational, levels. Different strategies are proposed to intervene to generate gene functional variations and consequently phenotype changes. For non-coding genes, trait modification could be achieved by regulating transcription of their own or target genes via gene editing. Also included is a scheme of protoplast editing to make gene editing more applicable in plant breeding. In summary, this review provides breeders with a host of options to translate gene biology into practical breeding strategies, i.e., to use gene editing as a mechanism to commercialize gene biology in plant breeding.展开更多
Biotechnology policies and regulations must be revised and updated to reflect the most recent advances in plantbreeding technology. New Plant Breeding Techniques(NPBT) such as gene editing have been applied to address...Biotechnology policies and regulations must be revised and updated to reflect the most recent advances in plantbreeding technology. New Plant Breeding Techniques(NPBT) such as gene editing have been applied to address the myriad of challenges in plant breeding, while the use of NPBT as emerging biotechnological tools raises legal and ethical concerns. This study aims to highlight how gene editing is operationalized in the existing literature and examine the critical issues of ethical and legal issues of gene editing for plant breeding. We carried out a systematic literature review(SLR) to provide the current states of ethical and legal discourses surrounding this topic. We also identified critical research priority areas and policy gaps that must be addressed when designing the future governance of gene editing in plant breeding.展开更多
The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise. Gene editing holds tremendous potential for furthe...The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise. Gene editing holds tremendous potential for further improvements of T cell therapy. Here we review the applications of gene editing in various T cell therapies, focusing on antiviral strategies and cancer immunotherapies, and discuss the challenges and future prospects.展开更多
Gene therapy has shown significant potential in treating various diseases,particularly inherited blood disorders such as hemophilia,sickle cell disease,and thalassemia.Advances in understanding the regulatory network ...Gene therapy has shown significant potential in treating various diseases,particularly inherited blood disorders such as hemophilia,sickle cell disease,and thalassemia.Advances in understanding the regulatory network of disease-associated genes have led to the identification of additional therapeutic targets for treatment,especially for β-hemoglobinopathies.Erythroid regulatory factor BCL11A offers the most promising therapeutic target for β-hemoglobinopathies,and reduction of its expression using the commercialized gene therapy product Casgevy has been approved for use in the UK and USA in 2023.Notably,the emergence of innovative gene editing technologies has further broadened the gene therapy landscape,presenting possibilities for treatment.Intensive studies indicate that base editing and prime editing,built upon CRISPR technology,enable precise single-base modification in hematopoietic stem cells for addressing inherited blood disorders ex vivo and in vivo.In this review,we present an overview of the current landscape of gene therapies,focusing on clinical research and gene therapy products for inherited blood disorders,evaluation of potential gene targets,and the gene editing tools employed in current gene therapy practices,which provides an insight for the establishment of safer and more effective gene therapy methods for a wider range of diseases in the future.展开更多
The typeⅡ prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR/Cas9) adaptive immune system is a cutting-edge genome-editing toolbox.However,its applications are still limited b...The typeⅡ prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR/Cas9) adaptive immune system is a cutting-edge genome-editing toolbox.However,its applications are still limited by its inefficient transduction.Herein,we present a novel gene vector,the zwitterionic polymer-inspired material with branched structure (ZEBRA) for efficient CRISPR/Cas9 delivery.Polo-like kinase 1 (PLK1) acts as a master regulator of mitosis and overexpresses in multiple tumor cells.The Cas9 and single guide sgRNA (sgRNA)-encoded plasmid was transduced to knockout Plk1 gene,which was expected to inhibit the expression of PLK1.Our studies demonstrated that ZEBRA enabled to transduce the CRISPR/Cas9 system with large size into the cells efficiently.The transduction with ZEBRA was cell line dependent,which showed~10-fold higher in CD44-positive cancer cell lines compared with CD44-negative ones.Furthermore,ZEBRA induced highlevel expression of Cas9 proteins by the delivery of CRISPR/Cas9 and efficient gene editing of Plk1 gene,and inhibited the tumor cell growth significantly.This zwitterionic polymerinspired material is an effective and targeted gene delivery vector and further studies are required to explore its potential in gene delivery applications.展开更多
The discovery and utilization of RNA-guided surveillance complexes,such as CRISPR-Cas9,for sequencespecific DNA or RNA cleavage,has revolutionised the process of gene modification or knockdown.To optimise the use of t...The discovery and utilization of RNA-guided surveillance complexes,such as CRISPR-Cas9,for sequencespecific DNA or RNA cleavage,has revolutionised the process of gene modification or knockdown.To optimise the use of this technology,an exploratory race has ensued to discover or develop new RNA-guided endonucleases with the most flexible sequence targeting requirements,coupled with high cleavage efficacy and specificity.Here we review the constraints of existing gene editing and assess the merits of exploiting the diversity of CRISPR-Cas effectors as a methodology for surmounting these limitations.展开更多
With advancements in gene editing technologies,our ability to make precise and efficient modifications to the genome is increasing at a remarkable rate,paving the way for scientists and clinicians to uniquely treat a ...With advancements in gene editing technologies,our ability to make precise and efficient modifications to the genome is increasing at a remarkable rate,paving the way for scientists and clinicians to uniquely treat a multitude of previously irremediable diseases.CRISPR-Cas9,short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9,is a gene editing platform with the ability to alter the nucleotide sequence of the genome in living cells.This technology is increasing the number and pace at which new gene editing treatments for genetic disorders are moving toward the clinic.Theβ-hemoglobinopathies are a group of monogenic diseases,which despite their high prevalence and chronic debilitating nature,continue to have few therapeutic options available.In this review,we will discuss our existing comprehension of the genetics and current state of treatment forβ-hemoglobinopathies,consider potential genome editing therapeutic strategies,and provide an overview of the current state of clinical trials using CRISPR-Cas9 gene editing.展开更多
The advent of gene editing represents one of the most transformative breakthroughs in life science,making genome manipulation more accessible than ever before.While traditional CRISPR/Cas-based gene editing,which invo...The advent of gene editing represents one of the most transformative breakthroughs in life science,making genome manipulation more accessible than ever before.While traditional CRISPR/Cas-based gene editing,which involves double-strand DNA breaks(DSBs),excels at gene disruption,it is less effective for accurate gene modification.The limitation arises because DSBs are primarily repaired via non-homologous end joining(NHEJ),which tends to introduce indels at the break site.While homology directed repair(HDR)can achieve precise editing when a donor DNA template is provided,the reliance on DSBs often results in unintended genome damage.HDR is restricted to specific cell cycle phases,limiting its application.Currently,gene editing has evolved to unprecedented levels of precision without relying on DSB and HDR.The development of innovative systems,such as base editing,prime editing,and CRISPR-associated transposases(CASTs),now allow for precise editing ranging from single nucleotides to large DNA fragments.Base editors(BEs)enable the direct conversion of one nucleotide to another,and prime editors(PEs)further expand gene editing capabilities by allowing for the insertion,deletion,or alteration of small DNA fragments.The CAST system,a recent innovation,allows for the precise insertion of large DNA fragments at specific genomic locations.In recent years,the optimization of these precise gene editing tools has led to significant improvements in editing efficiency,specificity,and versatility,with advancements such as the creation of base editors for nucleotide transversions,enhanced prime editing systems for more efficient and precise modifications,and refined CAST systems for targeted large DNA insertions,expanding the range of applications for these tools.Concurrently,these advances are complemented by significant improvements in in vivo delivery methods,which have paved the way for therapeutic application of precise gene editing tools.Effective delivery systems are critical for the success of gene therapies,and recent developments in both viral and non-viral vectors have improved the efficiency and safety of gene editing.For instance,adeno-associated viruses(AAVs)are widely used due to their high transfection efficiency and low immunogenicity,though challenges such as limited cargo capacity and potential for immune responses remain.Non-viral delivery systems,including lipid nanoparticles(LNPs),offer an alternative with lower immunogenicity and higher payload capacity,although their transfection efficiency can be lower.The therapeutic potential of these precise gene editing technologies is vast,particularly in treating genetic disorders.Preclinical studies have demonstrated the effectiveness of base editing in correcting genetic mutations responsible for diseases such as cardiomyopathy,liver disease,and hereditary hearing loss.These technologies promise to treat symptoms and potentially cure the underlying genetic causes of these conditions.Meanwhile,challenges remain,such as optimizing the safety and specificity of gene editing tools,improving delivery systems,and overcoming off-target effects,all of which are critical for their successful application in clinical settings.In summary,the continuous evolution of precise gene editing technologies,combined with advancements in delivery systems,is driving the field toward new therapeutic applications that can potentially transform the treatment of genetic disorders by targeting their root causes.展开更多
The molecular scalpel of clustered regularly interspersed short palindromic repeats/CRISPR associated protein 9(CRISPR/Cas9) technology may be sharp enough to begin cutting the genes implicated in inflammatory bowel d...The molecular scalpel of clustered regularly interspersed short palindromic repeats/CRISPR associated protein 9(CRISPR/Cas9) technology may be sharp enough to begin cutting the genes implicated in inflammatory bowel disease(IBD) and consequently decrease the 6.3 billion dollar annual financial healthcare burden in the treatment of IBD. For the past few years CRISPR technology has drastically revolutionized DNA engineering and biomedical research field. We are beginning to see its application in gene manipulation of sickle cell disease,human immunodeficiency virus resistant embryologic twin gene modification and IBD genes such as Gatm(Glycine amidinotransferase, mitochondrial),nucleotide-binding oligomerization domain-containing protein 2, KRT12 and other genes implicated in adaptive immune convergence pathways have been subjected to gene editing, however there are very few publications. Furthermore,since Crohn's disease and ulcerative colitis have shared disease susceptibility and share genetic gene profile, it is paramount and is more advantageous to use CRISPR technology to maximize impact. Although, currently CRISPR does have its limitations due to limited number of specific Cas enzymes, off-target activity,protospacer adjacent motifs and crossfire between different target sites. However,these limitations have given researchers further insight on how to augment and manipulate enzymes to enable precise gene excision and limit crossfire between target sites.展开更多
Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading...Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading to the development of clustered regularly interspaced short palindromic repeats(CRISPRs) and CRISPR-associated systems,zinc finger nucleases and transcription activator like effector nucleases have ushered in a new era for high throughput in vitro and in vivo genome engineering.Genome editing can be successfully used to decipher complex molecular mechanisms underlying disease pathophysiology,develop innovative next generation gene therapy,stem cell-based regenerative therapy,and personalized medicine for corneal and other ocular diseases.In this review we describe latest developments in the field of genome editing,current challenges,and future prospects for the development of personalized genebased medicine for corneal diseases.The gene editing approach is expected to revolutionize current diagnostic and treatment practices for curing blindness.展开更多
Correction of genetic errors, commonly known as gene editing, holds promise to treat diseases with unmet medical needs. However, gene therapy trials do encounter unwanted outcomes, because of an incomplete understandi...Correction of genetic errors, commonly known as gene editing, holds promise to treat diseases with unmet medical needs. However, gene therapy trials do encounter unwanted outcomes, because of an incomplete understanding of the disease states, and gene therapy processes, among others. This situation encourages a concept that healthcare professionals receiving laboratory research training will not only identify inadequacies in basic biomedical knowledge of gene therapies but also provide tangible refinements. To this end, we have undertaken the PharmD student training in gene editing in a basic research laboratory setting. As a model, MYC gene was chosen for knockout using CRISPR-Cas9 method in HT29 and OVCAR8 cells. Students were involved in the design of MYC-specific gRNAs, subcloning into Cas9-carrying plasmid, and selection of knockout clones from the transfected cells. Subsequently, genomic DNA isolation and sequencing, analysis of clonal DNA sequences using online bioinformatics tools, western blotting, cell proliferation and cell division cycle experiments, were performed to characterize the MYC knockout clones. Results presented in this communication suggest that healthcare professionals who received laboratory training gain a better understanding of the disease states and mechanisms, gene therapy protocols, limitations of gene therapies, ability to critically evaluate the literature and confidence in the oversight of gene therapies in the clinic.展开更多
Background:Corneal diseases are a major cause of global visual impairment,and current treatments remain inadequate for severe or refractory cases.The CRISPR/Cas system offers robust and precise gene-editing capabiliti...Background:Corneal diseases are a major cause of global visual impairment,and current treatments remain inadequate for severe or refractory cases.The CRISPR/Cas system offers robust and precise gene-editing capabilities,yet its therapeutic potential for corneal disorders remains largely unexplored.-Main text:This narrative review introduces the CRISPR/Cas system and summarizes its recent advances in treating various corneal diseases,including inherited corneal dystrophies,infectious keratitis,corneal injury,and pathological neovascularization.We outline emerging preclinical and clinical studies,and analyze key issues that should be addressed for translation,including administration strategies,vector platform optimization and the mitigation of off-target toxicity.Conclusions:This review provides a comprehensive and integrated overview of the current translational directions and challenges of CRISPR/Cas technology in corneal diseases from a novel perspective.It offers valuable guidance for future research and may accelerate the development of gene-editing therapies toward clinical application.展开更多
基金supported by Funding was provided by grants from the Changsha Chinese Medicine Foundation(Grant No.B202314)the Natural Science Foundation of Hunan Province,China(Grant No.2024JJ8224)+1 种基金Changsha Municipal Natural Science Foundation(Grant No.kq2403187)Hunan Province Children’s Safe Medication Clinical Medical Technology Demonstration Base(Grant No.2023SK4083).
文摘Pediatric congenital heart disease(CHD)pharmacotherapy faces three fundamental barriers:developmental pharmacokinetic complexity,anatomic-genetic heterogeneity,and evidence chain gaps.Traditional agents exhibit critical limitations:digoxin’s narrow therapeutic index(0.5–0.9 ng/mL)is exacerbated by ABCB1 mutations(toxicity risk increases 4.1-fold),furosemide efficacy declines by 35%in neonates due to NKCC2 immaturity,andβ-blocker responses vary by CYP2D6 polymorphisms(poor metabolizers require 50–75%dose reduction).Novel strategies demonstrate transformative potential—CRISPR editing achieves 81%reversal of BMPR2-associated pulmonary vascular remodeling,metabolically matured cardiac organoids replicate adult myocardial energy metabolism for drug screening,and SGLT2 inhibitors activate triple mechanisms(calcium overload mitigation,mitophagy,fibrosis reversal).However,clinical translation requires overcoming developmental barriers:age-dependent enzyme expression(infant CYP2D6=30–60%adult activity),post-Fontan hepatotoxicity(bosentan trough concentrations elevates 1.8-fold),and AI model limitations(32%error in complex CHD).Future integration of placental transfer models,disease-specific organoids,and multi-omics mapping of FOXO/CRIM1 pathways will shift paradigms from symptom control to curative repair.
基金supported by Jilin Provincial Department of Education(JKH20230394KJ).
文摘Clustered regularly interspaced short palindromic repeat sequences(CRISPR)and their accompanying proteins(Cas),commonly presenting in bacteria and archaea,make up the CRISPR/Cas system.As one of the funda-mental sources of nutrition for humans,edible crops play a crucial role in ensuring global food security.CRISPR/Cas9 gene editing has been applied to improve many crop traits,such as increasing nitrogen utilization efficiency,creating male sterile germplasm,and regulating tiller and spikelet formation.This paper provides a comprehensive overview of the use of CRISPR/Cas gene editing technology in crop genomes,covering the targeted genes,the types of editing that take place,the mechanism of action.Finally,we also discussed the efficiency of gene editing and pointed the future direction on how to speed up crop molecular breeding,increase breeding effectiveness,and produce more new crop varieties with high qualities.
基金supported by the National Institutes of Health(UH3NS115598).
文摘The potential of regenerative medicine in the clinical space is vast,given its ability to repair and replace damaged tissues,restore lost functions due to age or disease,and transform personalized therapy.Traditional regenerative medicine and tissue engineering strategies have created specialized tissues using progenitor cells and various biological stimuli.To date,there are many US Food and Drug Administration(FDA)-approved regenerative medicine therapies,such as those for wound healing and orthopedic injuries.Nonetheless,these therapies face challenges,including off-target effects,a lack of precision,and failure to target the disease or injury at its origin.In search of novel,precise,and efficient alternatives,the regenerative medicine landscape is shifting towards genome engineering technologies,particularly gene editing.Clustered regularly interspaced short palindromic repeats(CRISPR)-based gene editing systems enable precise knock-ins,knockouts,transcriptional activation and repression,as well as specific base conversions.This advancement has allowed researchers to treat genetic and degenerative diseases,control cell fate for highly regulated tissue repair,and enhance tissue functions.In this review,we explore the progress and future prospects of CRISPR technologies in regenerative medicine,focusing on how gene editing has led to advanced therapeutic applications and served as a versatile research tool for understanding tissue development and disease progression.
文摘Nonobstructive azoospermia(NOA)affects about 60%of men with azoospermia,representing a severe form of male infertility.The current approach to manage NOA primarily involves testicular sperm retrieval methods such as conventional testicular sperm extraction(c-TESE)and microdissection testicular sperm extraction(micro-TESE).While combining testicular sperm retrieval with intracytoplasmic sperm injection(ICSI)offers hope for patients,the overall sperm retrieval rate(SRR)stands at around 50%.In cases where micro-TESE fails to retrieve sperm,limited options,like donor sperm or adoption,can be problematic in certain cultural contexts.This paper delves into prospective treatments for NOA management.Gene editing technologies,particularly clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)protein 9(CRISPR/Cas9),hold potential for correcting genetic mutations underlying testicular dysfunction.However,these technologies face challenges due to their complexity,potential off-target effects,ethical concerns,and affordability.This calls for research to address key challenges associated with NOA management within the clinical settings.This also necessitate ongoing research essential for developing more sensitive diagnostic tests,validating novel treatments,and customizing current treatment strategies for individual patients.This review concluded that the future of NOA management may entail a combination of these treatment options,tailored to each patient’s unique circumstances,providing a comprehensive approach to address NOA challenges.
基金This study was supported by the National Natural Science Foundation of China(Nos.31771648 and 31672653)the Scientific Research Foundation of Jimei University(No.ZQ2020003)the National Key Basic Research Program of China(No.2013CB967700).
文摘Generation of mutants with clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA(mRNA)or protein and transcribed guide RNA(gRNA).However,the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present.In this study,we employed a poly-transfer RNA(tRNA)-gRNA(PTG)system driven by cytomegalovirus(CMV)promoter to target the medaka(Oryzias latipes)endogenous gene tyrosinase(tyr)or paired box 6.1(pax6.1)and illustrated its function in a medaka cell line and embryos.The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka.This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.
基金supported by the Ministry of Science and Technology of China (2014CB964803 and 2015AA020307)the National Natural Science Foundation of China (Nos. 31530048, 31601163 and 81672117)+1 种基金he Chinese Academy of Sciences (XDB19010204 and QYZDJ-SSW-SMC023)the Shanghai Municipal Commission for Science and Technology(16JC1420500, 17JC1400900 and 17140901500)
文摘Generation of mouse models carrying a defined point mutation,especially disease-related point mutations,is of considerable interest for research in biology and medicine.The standard method based on embryonic stem cell（ESC）-mediated homologous recombination（HR）is time-and labor-consuming.
基金by grants from the National Research Foundation,Ministry of Science and ICT,Republic of Korea(NRF-2021R1A2B5B03002123,NRF-2018R1A5A2024425,NRF-2021K2A9A2A06044515,2022M3E5F1017919)Ministry of Education,Republic of Korea(NRF-2021R1A6A3A01086428)Korean Health Technology R&D Project(No.HI19C0664),Ministry of Health&Welfare,Republic of Korea.
文摘Metabolic engineering of the tumor microenvironment has emerged as a new strategy.Lactate dehydrogenase A(LDHA)is a prominent target for metabolic engineering.Here,we designed a cationic lipid nanoparticle formulation for LDHA gene editing.The plasmid DNA delivery efficiency of our lipid nanoparticle formulations was screened by testing the fluorescence of lipid nanoparticles complexed to plasmid DNA encoding green fluorescence protein(GFP).The delivery efficiency was affected by the ratios of three components:a cationic lipid,cholesterol or its derivative,and a fusogenic lipid.The lipid nanoparticle designated formulation F3 was complexed to plasmid DNA co-encoding CRISPR-associated protein 9 and LDHA-specific sgRNA,yielding the lipoplex,pCas9-sgLDHA/F3.The lipoplex including GFP-encoding plasmid DNA provided gene editing in HeLa-GFP cells.Treatment of B16F10 tumor cells with pCas9-sgLDHA/F3 yielded editing of the LDHA gene and increased the pH of the culture medium.pCas9-sgLDHA/F3 treatment activated the interferon-gamma and granzyme production of T cells in culture.In vivo,combining pCas9-sgLDHA/F3 with immune checkpoint-inhibiting anti-PD-L1 antibody provided a synergistic antitumor effect and prolonged the survival of tumor model mice.This study suggests that combining metabolic engineering of the tumor microenvironment with immune checkpoint inhibition could be a valuable antitumor strategy.
基金supported by the Fundamental Research Funds for the Central Universities(2042021kf0195)love creates future research funding,and Hubei natural science foundation(2021CFB483)+2 种基金supported by grants from China National Special Program for Major Infectious Diseases(2014ZX10001003 and 2017ZX10202102)the National Natural Science Foundation of China(81401659,82172258)China Postdoctoral Science Foundation(2015T80838 and 2014M560622)
文摘Although tremendous efforts have been made to prevent and treat HIV-1 infection,HIV-1/AIDS remains a major threat to global human health.The combination antiretroviral therapy(cART),although able to suppress HIV-1 replication,cannot eliminate the proviral DNA integrated into the human genome and thus requires lifelong treatment that may lead to various side effects.In recent years,clustered regularly interspaced short palindromic repeat(CRISPR)-associated nuclease 9(Cas9)related gene-editing systems have been developed and designed as effective ways to treat HIV-1 infection.However,new gene-targeting tools derived from or functioning like CRISPR/Cas9,including base editor,prime editing,SHERLOCK,DETECTR,PAC-MAN,ABACAS,pfAGO,have been developed and optimized for pathogens detection and diseases correction.Here,we summarize recent studies on HIV-1/AIDS gene therapy and provide more gene-editing targets based on studies relating to the molecular mechanism of HIV-1 infection.We also identify the strategies and potential applications of these new gene-editing technologies for HIV-1/AIDS treatment in the future.Moreover,we discuss the caveats and problems that should be addressed before the clinical use of these versatile CRISPR-based gene targeting tools.Finally,we offer alternative solutions to improve the practice of gene targeting in HIV-1/AIDS gene therapy.
基金Project supported by the Zhejiang Provincial S&T Project on Breeding Agricultural(Food)Crops(No.2016C02050-2)the National Natural Science Foundation of China(No.31701394)。
文摘Plant breeding is well recognized as one of the most important means to meet food security challenges caused by the ever-increasing world population. During the past three decades, plant breeding has been empowered by both new knowledge on trait development and regulation(e.g., functional genomics) and new technologies(e.g., biotechnologies and phenomics). Gene editing, particularly by clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas) and its variants, has become a powerful technology in plant research and may become a game-changer in plant breeding. Traits are conferred by coding and non-coding genes. From this perspective, we propose different editing strategies for these two types of genes. The activity of an encoded enzyme and its quantity are regulated at transcriptional and post-transcriptional, as well as translational and post-translational, levels. Different strategies are proposed to intervene to generate gene functional variations and consequently phenotype changes. For non-coding genes, trait modification could be achieved by regulating transcription of their own or target genes via gene editing. Also included is a scheme of protoplast editing to make gene editing more applicable in plant breeding. In summary, this review provides breeders with a host of options to translate gene biology into practical breeding strategies, i.e., to use gene editing as a mechanism to commercialize gene biology in plant breeding.
基金supported by the Ministry of Higher Education(MoHE)Malaysia under the Fundamental Research Grant Scheme(No.FRGS/1/2021/SS0/UM/02/6)the Universiti Malaya Research University Grant(No.RU004A-2020).
文摘Biotechnology policies and regulations must be revised and updated to reflect the most recent advances in plantbreeding technology. New Plant Breeding Techniques(NPBT) such as gene editing have been applied to address the myriad of challenges in plant breeding, while the use of NPBT as emerging biotechnological tools raises legal and ethical concerns. This study aims to highlight how gene editing is operationalized in the existing literature and examine the critical issues of ethical and legal issues of gene editing for plant breeding. We carried out a systematic literature review(SLR) to provide the current states of ethical and legal discourses surrounding this topic. We also identified critical research priority areas and policy gaps that must be addressed when designing the future governance of gene editing in plant breeding.
基金supported by the National Natural Science Foundation of China (No. 31471215)the National High-tech R&D Program (863 Program) (2015AA020307)supported by the "Young Thousand Talent Project"
文摘The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise. Gene editing holds tremendous potential for further improvements of T cell therapy. Here we review the applications of gene editing in various T cell therapies, focusing on antiviral strategies and cancer immunotherapies, and discuss the challenges and future prospects.
基金supported by Research Fund of Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital(30420230006)the National Natural Science Foundation of China(Nos.82300142)Sichuan Science and Technology Program(2022ZYD0131)。
文摘Gene therapy has shown significant potential in treating various diseases,particularly inherited blood disorders such as hemophilia,sickle cell disease,and thalassemia.Advances in understanding the regulatory network of disease-associated genes have led to the identification of additional therapeutic targets for treatment,especially for β-hemoglobinopathies.Erythroid regulatory factor BCL11A offers the most promising therapeutic target for β-hemoglobinopathies,and reduction of its expression using the commercialized gene therapy product Casgevy has been approved for use in the UK and USA in 2023.Notably,the emergence of innovative gene editing technologies has further broadened the gene therapy landscape,presenting possibilities for treatment.Intensive studies indicate that base editing and prime editing,built upon CRISPR technology,enable precise single-base modification in hematopoietic stem cells for addressing inherited blood disorders ex vivo and in vivo.In this review,we present an overview of the current landscape of gene therapies,focusing on clinical research and gene therapy products for inherited blood disorders,evaluation of potential gene targets,and the gene editing tools employed in current gene therapy practices,which provides an insight for the establishment of safer and more effective gene therapy methods for a wider range of diseases in the future.
基金National Natural Science Foundation of China(82072047,81700382)Natural Science Foundation of Guangdong Province(2019A1515012166)+2 种基金Research Foundation of Education Bureau of Guangdong Province(2021ZDZX2004)Basic and Applied Basic Research Project of Guangzhou(02080390)Outstanding Youth Development Program of Guangzhou Medical University.
文摘The typeⅡ prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR/Cas9) adaptive immune system is a cutting-edge genome-editing toolbox.However,its applications are still limited by its inefficient transduction.Herein,we present a novel gene vector,the zwitterionic polymer-inspired material with branched structure (ZEBRA) for efficient CRISPR/Cas9 delivery.Polo-like kinase 1 (PLK1) acts as a master regulator of mitosis and overexpresses in multiple tumor cells.The Cas9 and single guide sgRNA (sgRNA)-encoded plasmid was transduced to knockout Plk1 gene,which was expected to inhibit the expression of PLK1.Our studies demonstrated that ZEBRA enabled to transduce the CRISPR/Cas9 system with large size into the cells efficiently.The transduction with ZEBRA was cell line dependent,which showed~10-fold higher in CD44-positive cancer cell lines compared with CD44-negative ones.Furthermore,ZEBRA induced highlevel expression of Cas9 proteins by the delivery of CRISPR/Cas9 and efficient gene editing of Plk1 gene,and inhibited the tumor cell growth significantly.This zwitterionic polymerinspired material is an effective and targeted gene delivery vector and further studies are required to explore its potential in gene delivery applications.
基金the National Health and Medical Research Council of Australia(Grant No.APP1143008)the Australian Research Council(Grant No.DP180101494)the National Natural Science Foundation of China(Grant No.81772214).
文摘The discovery and utilization of RNA-guided surveillance complexes,such as CRISPR-Cas9,for sequencespecific DNA or RNA cleavage,has revolutionised the process of gene modification or knockdown.To optimise the use of this technology,an exploratory race has ensued to discover or develop new RNA-guided endonucleases with the most flexible sequence targeting requirements,coupled with high cleavage efficacy and specificity.Here we review the constraints of existing gene editing and assess the merits of exploiting the diversity of CRISPR-Cas effectors as a methodology for surmounting these limitations.
文摘With advancements in gene editing technologies,our ability to make precise and efficient modifications to the genome is increasing at a remarkable rate,paving the way for scientists and clinicians to uniquely treat a multitude of previously irremediable diseases.CRISPR-Cas9,short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9,is a gene editing platform with the ability to alter the nucleotide sequence of the genome in living cells.This technology is increasing the number and pace at which new gene editing treatments for genetic disorders are moving toward the clinic.Theβ-hemoglobinopathies are a group of monogenic diseases,which despite their high prevalence and chronic debilitating nature,continue to have few therapeutic options available.In this review,we will discuss our existing comprehension of the genetics and current state of treatment forβ-hemoglobinopathies,consider potential genome editing therapeutic strategies,and provide an overview of the current state of clinical trials using CRISPR-Cas9 gene editing.
文摘The advent of gene editing represents one of the most transformative breakthroughs in life science,making genome manipulation more accessible than ever before.While traditional CRISPR/Cas-based gene editing,which involves double-strand DNA breaks(DSBs),excels at gene disruption,it is less effective for accurate gene modification.The limitation arises because DSBs are primarily repaired via non-homologous end joining(NHEJ),which tends to introduce indels at the break site.While homology directed repair(HDR)can achieve precise editing when a donor DNA template is provided,the reliance on DSBs often results in unintended genome damage.HDR is restricted to specific cell cycle phases,limiting its application.Currently,gene editing has evolved to unprecedented levels of precision without relying on DSB and HDR.The development of innovative systems,such as base editing,prime editing,and CRISPR-associated transposases(CASTs),now allow for precise editing ranging from single nucleotides to large DNA fragments.Base editors(BEs)enable the direct conversion of one nucleotide to another,and prime editors(PEs)further expand gene editing capabilities by allowing for the insertion,deletion,or alteration of small DNA fragments.The CAST system,a recent innovation,allows for the precise insertion of large DNA fragments at specific genomic locations.In recent years,the optimization of these precise gene editing tools has led to significant improvements in editing efficiency,specificity,and versatility,with advancements such as the creation of base editors for nucleotide transversions,enhanced prime editing systems for more efficient and precise modifications,and refined CAST systems for targeted large DNA insertions,expanding the range of applications for these tools.Concurrently,these advances are complemented by significant improvements in in vivo delivery methods,which have paved the way for therapeutic application of precise gene editing tools.Effective delivery systems are critical for the success of gene therapies,and recent developments in both viral and non-viral vectors have improved the efficiency and safety of gene editing.For instance,adeno-associated viruses(AAVs)are widely used due to their high transfection efficiency and low immunogenicity,though challenges such as limited cargo capacity and potential for immune responses remain.Non-viral delivery systems,including lipid nanoparticles(LNPs),offer an alternative with lower immunogenicity and higher payload capacity,although their transfection efficiency can be lower.The therapeutic potential of these precise gene editing technologies is vast,particularly in treating genetic disorders.Preclinical studies have demonstrated the effectiveness of base editing in correcting genetic mutations responsible for diseases such as cardiomyopathy,liver disease,and hereditary hearing loss.These technologies promise to treat symptoms and potentially cure the underlying genetic causes of these conditions.Meanwhile,challenges remain,such as optimizing the safety and specificity of gene editing tools,improving delivery systems,and overcoming off-target effects,all of which are critical for their successful application in clinical settings.In summary,the continuous evolution of precise gene editing technologies,combined with advancements in delivery systems,is driving the field toward new therapeutic applications that can potentially transform the treatment of genetic disorders by targeting their root causes.
文摘The molecular scalpel of clustered regularly interspersed short palindromic repeats/CRISPR associated protein 9(CRISPR/Cas9) technology may be sharp enough to begin cutting the genes implicated in inflammatory bowel disease(IBD) and consequently decrease the 6.3 billion dollar annual financial healthcare burden in the treatment of IBD. For the past few years CRISPR technology has drastically revolutionized DNA engineering and biomedical research field. We are beginning to see its application in gene manipulation of sickle cell disease,human immunodeficiency virus resistant embryologic twin gene modification and IBD genes such as Gatm(Glycine amidinotransferase, mitochondrial),nucleotide-binding oligomerization domain-containing protein 2, KRT12 and other genes implicated in adaptive immune convergence pathways have been subjected to gene editing, however there are very few publications. Furthermore,since Crohn's disease and ulcerative colitis have shared disease susceptibility and share genetic gene profile, it is paramount and is more advantageous to use CRISPR technology to maximize impact. Although, currently CRISPR does have its limitations due to limited number of specific Cas enzymes, off-target activity,protospacer adjacent motifs and crossfire between different target sites. However,these limitations have given researchers further insight on how to augment and manipulate enzymes to enable precise gene excision and limit crossfire between target sites.
文摘Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading to the development of clustered regularly interspaced short palindromic repeats(CRISPRs) and CRISPR-associated systems,zinc finger nucleases and transcription activator like effector nucleases have ushered in a new era for high throughput in vitro and in vivo genome engineering.Genome editing can be successfully used to decipher complex molecular mechanisms underlying disease pathophysiology,develop innovative next generation gene therapy,stem cell-based regenerative therapy,and personalized medicine for corneal and other ocular diseases.In this review we describe latest developments in the field of genome editing,current challenges,and future prospects for the development of personalized genebased medicine for corneal diseases.The gene editing approach is expected to revolutionize current diagnostic and treatment practices for curing blindness.
文摘Correction of genetic errors, commonly known as gene editing, holds promise to treat diseases with unmet medical needs. However, gene therapy trials do encounter unwanted outcomes, because of an incomplete understanding of the disease states, and gene therapy processes, among others. This situation encourages a concept that healthcare professionals receiving laboratory research training will not only identify inadequacies in basic biomedical knowledge of gene therapies but also provide tangible refinements. To this end, we have undertaken the PharmD student training in gene editing in a basic research laboratory setting. As a model, MYC gene was chosen for knockout using CRISPR-Cas9 method in HT29 and OVCAR8 cells. Students were involved in the design of MYC-specific gRNAs, subcloning into Cas9-carrying plasmid, and selection of knockout clones from the transfected cells. Subsequently, genomic DNA isolation and sequencing, analysis of clonal DNA sequences using online bioinformatics tools, western blotting, cell proliferation and cell division cycle experiments, were performed to characterize the MYC knockout clones. Results presented in this communication suggest that healthcare professionals who received laboratory training gain a better understanding of the disease states and mechanisms, gene therapy protocols, limitations of gene therapies, ability to critically evaluate the literature and confidence in the oversight of gene therapies in the clinic.
基金supported by the National Natural Science Foundation of China(No.82571186,82371037,82171033)the National Key R&D Program of China(No.2024YFC2510900/2024YFC2510904).
文摘Background:Corneal diseases are a major cause of global visual impairment,and current treatments remain inadequate for severe or refractory cases.The CRISPR/Cas system offers robust and precise gene-editing capabilities,yet its therapeutic potential for corneal disorders remains largely unexplored.-Main text:This narrative review introduces the CRISPR/Cas system and summarizes its recent advances in treating various corneal diseases,including inherited corneal dystrophies,infectious keratitis,corneal injury,and pathological neovascularization.We outline emerging preclinical and clinical studies,and analyze key issues that should be addressed for translation,including administration strategies,vector platform optimization and the mitigation of off-target toxicity.Conclusions:This review provides a comprehensive and integrated overview of the current translational directions and challenges of CRISPR/Cas technology in corneal diseases from a novel perspective.It offers valuable guidance for future research and may accelerate the development of gene-editing therapies toward clinical application.
