Rice, a global staple food, is critical for food security. The cultivated Oryza sativa, domesticated from wild O. rufipogon, derives~80%of its 993 identified domestication-related genes from O. rufipogon and 20%from S...Rice, a global staple food, is critical for food security. The cultivated Oryza sativa, domesticated from wild O. rufipogon, derives~80%of its 993 identified domestication-related genes from O. rufipogon and 20%from South/Southeast Asian wild O. nivara(Jing et al., 2023). Genes like An-1, BH4, PROG1,SH4, Rc, Rd, and GS3—which regulate awn length, hull color,til er angle, seed shattering, pericarp color, seed length, and thousand-grain weight, respectively—were selected against during domestication to form modern O. sativa(Yu et al., 2021).However, domestication and yield-focused breeding eliminated wild rice's valuable genes(e.g., for disease resistance, stress tolerance, nutrition), narrowing genetic diversity and impeding efforts to meet growing societal demands.展开更多
Emerging and powerful genome editing tools,particularly CRISPR/Cas9,are facilitating functional genomics research and accelerating crop improvement(Jiang et al.2021;Cao et al.2023;Chen C et al.2023;Liu et al.2023a).Ho...Emerging and powerful genome editing tools,particularly CRISPR/Cas9,are facilitating functional genomics research and accelerating crop improvement(Jiang et al.2021;Cao et al.2023;Chen C et al.2023;Liu et al.2023a).However,the detection and screening of transgenic lines remain major bottlenecks,being time-consuming,labor-intensive,and inefficient during transformation and subsequent mutation identification.A simple and efficient visual marker system plays a critical role in addressing these challenges.Recent studies demonstrated that the GmW1 and RUBY reporter systems were used to obtain visual transgenic soybean(Glycine max) plants(Chen L et al.2023;Chen et al.2024).展开更多
Schizophrenia（SZ） is a devastating mental disorder afflicting 1% of the population. Recent genome-wide association studies（GWASs） of SZ have identified 〉100 risk loci. However,the causal variants/genes and the ca...Schizophrenia（SZ） is a devastating mental disorder afflicting 1% of the population. Recent genome-wide association studies（GWASs） of SZ have identified 〉100 risk loci. However,the causal variants/genes and the causal mechanisms remain largely unknown,which hinders the translation of GWAS fi ndings into disease biology and drug targets. Most risk variants are noncoding,thus likely regulate gene expression. A major mechanism of transcriptional regulation is chromatin remodeling,and open chromatin is a versatile predictor of regulatory sequences. Micro RNA-mediated post-transcriptional regulation plays an important role in SZ pathogenesis. Neurons differentiated from patient-specifi c induced pluripotent stem cells（i PSCs） provide an experimental model to characterize the genetic perturbation of regulatory variants that are often specifi c to cell type and/or developmental stage. The emerging genome-editing technology enables the creation ofisogenic i PSCs and neurons to effi ciently characterize the effects of SZ-associated regulatory variants on SZ-relevant molecular and cellular phenotypes involving dopaminergic,glutamatergic,and GABAergic neurotransmissions. SZ GWAS fi ndings equipped with the emerging functional genomics approaches provide an unprecedented opportunity for understanding new disease biology and identifying novel drug targets.展开更多
Genome editing in plants is a powerful strategy that can substantially advance functional genomics research,facilitating the discovery,enhancement,and development of novel traits with significant agricultural implicat...Genome editing in plants is a powerful strategy that can substantially advance functional genomics research,facilitating the discovery,enhancement,and development of novel traits with significant agricultural implications.Various methodologies,such as zinc finger nucleases(ZFNs),transcription activator-like effector nucleases(TALENs),and CRISPR/Cas systems,have been developed for this purpose.展开更多
Precise chromosome engineering has traditionally relied on the Cre-Lox recombination system-an approach in which the enzyme Cre functions like molecular scissors,cutting and rejoining DNA at specific“Lox”sites to ad...Precise chromosome engineering has traditionally relied on the Cre-Lox recombination system-an approach in which the enzyme Cre functions like molecular scissors,cutting and rejoining DNA at specific“Lox”sites to add,remove,or flip genomic DNA segments inside living cells.展开更多
CRISPR/Cas9 technology, a revolutionary gene-editing tool, has rapidly garnered attention in plant science owing to its simplicity, high editing efficiency, and cost-effectiveness. Besides, it offers unprecedented pre...CRISPR/Cas9 technology, a revolutionary gene-editing tool, has rapidly garnered attention in plant science owing to its simplicity, high editing efficiency, and cost-effectiveness. Besides, it offers unprecedented precision and efficiency in the genetic improvement of fruit trees. To date, this technology has been widely utilized to enhance fruit quality, improve stress resistance, and mediate growth and development. These applications demonstrate its immense potential in fruit tree breeding. Looking ahead, advancements in editing efficiency, expanded application scopes, comprehensive safety assessments, and improved regulatory frameworks are expected to further broaden the role of CRISPR/Cas9 in fruit tree breeding, thereby driving the fruit tree industry toward higher yield, superior quality, enhanced stress resilience, higher efficiency, and contributing to global food security and sustainable agricultural development. This article outlines the fundamental principles of CRISPR/Cas9 gene editing technology, its applications in plants (including fruit trees), and its pivotal role in genetic improvement and germplasm innovation.展开更多
Tomato,a vegetable of considerable global significance,has been the subject of extensive breeding efforts primarily aimed at achieving high yields and resistance to multiple stressors.However,despite these endeavors,t...Tomato,a vegetable of considerable global significance,has been the subject of extensive breeding efforts primarily aimed at achieving high yields and resistance to multiple stressors.However,despite these endeavors,the quality of fruits still cannot fully satisfy the diverse preferences of the majority of consumers.The elucidation of genetic determinants underlying fruit quality traits,coupled with the advancement of gene editing techniques,has significantly contributed to the enhancement of tomato quality.Combining with gene editing technology to improve tomato fruit quality traits represents a viable approach for maximizing the utilization of essential genes in breeding programs.This review provides a comprehensive summary of the significant genes associated with tomato fruit quality traits,as well as an overview of the current advancements and potential avenues for enhancing tomato quality through gene editing technology.Four important aspects of fruit quality-appearance,flavor,nutritional profiles and postharvest properties form the basis of the review,providing a thorough update on the state of research in tomato fruit quality improvement via new gene editing techniques.展开更多
Gastric cancer(GC)remains one of the leading causes of cancer-related mortality worldwide,necessitating innovative approaches for its diagnosis and treatment.Clustered regularly interspaced short palindromic repeats(C...Gastric cancer(GC)remains one of the leading causes of cancer-related mortality worldwide,necessitating innovative approaches for its diagnosis and treatment.Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPRassociated protein 9(Cas9),a revolutionary gene-editing technology,has emerged as a powerful tool for unraveling the molecular mechanisms underlying GC and for advancing precision medicine strategies.This review explores the current applications of CRISPR/Cas9 in GC research,including the identification of oncogenes and tumor suppressors,modeling tumor microenvironment interactions,and developing gene-based therapies.We highlight recent breakthroughs in genome editing that have enhanced our understanding of GC pathogenesis and resistance mechanisms to conventional therapies.Additionally,we discuss the potential of CRISPR/Cas9 for therapeutic gene editing in GC,addressing challenges such as off-target effects,delivery methods,and ethical considerations.By summarizing the progress and limitations of CRISPR/Cas9 in GC,this review aims to provide a comprehensive perspective on how this transformative technology could shape future strategies for the prevention,diagnosis,and treatment of GC.展开更多
Optimizing plant architecture for specific cultivation methods is essential for enhancing fruit productivity.Unlike indeterminate growth plants,the total productivity of determinate growth plants relies on cumulative ...Optimizing plant architecture for specific cultivation methods is essential for enhancing fruit productivity.Unlike indeterminate growth plants,the total productivity of determinate growth plants relies on cumulative fruit production and synchronized fruit ripening from both main and axillary shoots.Here,we focused on SlD14and SlMAX1,two key genes involved in the regulation of strigolactone(SL)signaling and biosynthesis,with the goal of maximizing yield and syn chronizing fruit ripening by fine-tuning axillary shoot growth.Using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)technology,we found that the sld14,slmax1,and sld14 slmax1mutant plants exhibited reduced plant height and increased axillary shoot proliferation compared to wild-type plants.However,these mutants showed reduced yield and delayed ripening,likely due to a source-sink imbalance caused by excessive axillary shoot development.A weak sld14 allele displayed a milder phenotype,maintaining total fruit yield and harvest index despite smaller individual fruit size.These findings indicate that allelic variation in SL-related genes can influence plant architecture and yield components.Our results suggest that weak or partial alleles may serve as promising targets for tailoring tomato architecture to space-limited cultivation systems.展开更多
Generating mutants bearing multiple gene modifications is essential for determining the functions of gene family members with redundant functions, or for analyzing epistatic re- lationships in genetic pathways. Using ...Generating mutants bearing multiple gene modifications is essential for determining the functions of gene family members with redundant functions, or for analyzing epistatic re- lationships in genetic pathways. Using conventional methods, mutants with multiple gene mutations are generated by several rounds of intercrossing plants carrying a single mutation and identification of the offspring. This process is both timeconsuming and labor-intensive. Moreover, if the genes of interest are closely linked, multiple mutations can not be generated （Wijnker and de Jong, 2008）.展开更多
Rice is a staple food for more than half of the human population.It has been estimated that by 2030,40%more rice needs to be produced in order to meet the growing demand（Khush,2005）.One of the strategies to improve ...Rice is a staple food for more than half of the human population.It has been estimated that by 2030,40%more rice needs to be produced in order to meet the growing demand（Khush,2005）.One of the strategies to improve rice productivity is to enlarge rice growth areas, such as the northward expansion of the growth region in Heilongjiang Province, the northernmost region of China （Li et al., 2015）. However, the northward cultivation is accompanied with daylength extension and temperature decrease, which are unfavor- able for rice, a tropical short-day plant, to complete flowering and seed setting. Thus, the need for early-maturing rice cultivars with extremely low photoperiod sensitivity is urgent.展开更多
Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010 s,clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)has ra...Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010 s,clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)has rapidly been developed into a robust,multifunctional genome editing tool with many uses.Following the discovery of the initial CRISPR/Cas-based system,the technology has been advanced to facilitate a multitude of different functions.These include development as a base editor,prime editor,epigenetic editor,and CRISPR interference(CRISPRi)and CRISPR activator(CRISPRa)gene regulators.It can also be used for chromatin and RNA targeting and imaging.Its applications have proved revolutionary across numerous biological fields,especially in biomedical and agricultural improvement.As a diagnostic tool,CRISPR has been developed to aid the detection and screening of both human and plant diseases,and has even been applied during the current coronavirus disease 2019(COVID-19)pandemic.CRISPR/Cas is also being trialed as a new form of gene therapy for treating various human diseases,including cancers,and has aided drug development.In terms of agricultural breeding,precise targeting of biological pathways via CRISPR/Cas has been key to regulating molecular biosynthesis and allowing modification of proteins,starch,oil,and other functional components for crop improvement.Adding to this,CRISPR/Cas has been shown capable of significantly enhancing both plant tolerance to environmental stresses and overall crop yield via the targeting of various agronomically important gene regulators.Looking to the future,increasing the efficiency and precision of CRISPR/Cas delivery systems and limiting off-target activity are two major challenges for wider application of the technology.This review provides an in-depth overview of current CRISPR development,including the advantages and disadvantages of the technology,recent applications,and future considerations.展开更多
Single-guide RNA(sg RNA) is one of the two core components of the CRISPR(clustered regularly interspaced short palindromic repeat)/Cas(CRISPR-associated) genome-editing technology. We established an in vitro Traffic L...Single-guide RNA(sg RNA) is one of the two core components of the CRISPR(clustered regularly interspaced short palindromic repeat)/Cas(CRISPR-associated) genome-editing technology. We established an in vitro Traffic Light Reporter(TLR) system, which is designated as the same colors as traffic lights such as green, red and yellow were produced in cells. The TLR can be readily used in maize mesophyll protoplast for a quick test of promoter activity. The TLR assay indicates the variation in transcription activities of the seven Pol III promoters, from 3.4%(U6-1) to over 21.0%(U6-6). The U6-2 promoter, which was constructed to drive sg RNA expression targeting the Zm Wx1 gene, yielded mutation efficiencies ranging from 48.5% to 97.1%. Based on the reported and unpublished data, the in vitro TLR assay results were confirmed to be a readily system and may be extended to other plant species amenable to efficient genome editing via CRISPR/Cas. Our efforts provide an efficient method of identifying native Pol III-recognized promoters for RNA guide-based genome-editing systems in maize.展开更多
Grain size is one of the most important factors affecting rice grain quality and yield,and attracts great attention from molecular biologists and breeders.In this study,we engineered a CRISPR/Cas9 system targeting the...Grain size is one of the most important factors affecting rice grain quality and yield,and attracts great attention from molecular biologists and breeders.In this study,we engineered a CRISPR/Cas9 system targeting the miR396 recognition site of the rice GS2 gene,which encodes growth-regulating factor 4(OsGRF4)and regulates multiple agronomic traits including grain size,grain quality,nitrogen use efficiency,abiotic stress response,and seed shattering.In contrast to most previous genome editing efforts in which indel mutations were chosen to obtain null mutants,a mutant named GS2^(E) carrying an in-frame 6-bp deletion and 1-bp substitution within the miR396-targeted sequence was identified.GS2^(E) plants showed increased expression of GS2 in consistent with impaired repression by miR396.As expected,the gain-of-function GS2^(E) mutant exhibited multiple beneficial traits including increased grain size and yield and bigger grain length/width ratio.Thousand grain weight and grain yield per plant of GS2^(E) plants were increased by 23.5%and 10.4%,respectively.These improved traits were passed to hybrids in a semidominant way,suggesting that the new GS2^(E) allele has great potential in rice improvement.Taken together,we report new GS2 germplasm and describe a novel gene-editing strategy that can be widely employed to improve grain size and yield in rice.This trait-improvement strategy could be applied to other genes containing miRNA target sites,in particular the conserved miR396-GRF/GIF module that governs plant growth,development and environmental response.展开更多
The quality traits of horticultural crops,including the accumulation of nutrients and flavor substances,morphology,and texture,affect the palatability and nutritional value.For many years,efforts have been made to imp...The quality traits of horticultural crops,including the accumulation of nutrients and flavor substances,morphology,and texture,affect the palatability and nutritional value.For many years,efforts have been made to improve the quality of horticultural crops.The recent establishment of gene editing technology,with its potential applications in horticultural crops,provides a strategy for achieving this goal in a rapid and efficient manner.Here,we summarize research efforts aimed at improving horticultural crop quality through genome editing.We describe specific genome editing systems that have been used and traits that have been targeted in these efforts.Additionally,we discuss limiting factors and future perspectives of genome editing technology in improving horticultural crop qualities in both research and plant breeding.In summary,genome editing technology is emerging as a powerful tool for efficiently and rapidly improving horticultural crop quality,and we believe that the cautious application of genome editing in horticultural crops will generate new germplasms with improved quality in the near future.展开更多
MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both acade...MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both academic and commercial use.Here,we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences(YTTN)in plants.Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-Lb Cas12 a system.We develop two variants,MAD7-RR and MAD7-RVR that increase the target range of MAD7,as well as an M-AFID(a MAD7-APOBEC fusion-induced deletion)system that creates predictable deletions from 50-deaminated Cs to the MAD7-cleavage site.Moreover,we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs.Using the CRISPR-MAD7 system,we have obtained regenerated mutant rice and wheat plants with up to 65.6%efficiency.展开更多
The last couple of years have witnessed an explosion in development of CRISPR-based genome editing technologies in cell lines as well as in model organisms. In this review, we focus on the applications of this popular...The last couple of years have witnessed an explosion in development of CRISPR-based genome editing technologies in cell lines as well as in model organisms. In this review, we focus on the applications of this popular system in Drosophila. We discuss the effectiveness of the CRISPR/Cas9 systems in terms of delivery, mutagenesis detection, parameters affecting efficiency, and off-target issues, with an emphasis on how to apply this powerful tool to characterize gene functions.展开更多
Development of tools for targeted modifications of specific DNA sequences in plants is of great importance to basic plant biology research as well as crop improvement.The ability to cut DNA at specific locations in th...Development of tools for targeted modifications of specific DNA sequences in plants is of great importance to basic plant biology research as well as crop improvement.The ability to cut DNA at specific locations in the genome to generate doublestrand breaks（DSBs）in vivo is a prerequisite for any genome editing efforts.展开更多
Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression sys...Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.展开更多
Prokaryotic type II adaptive immune systems have been developed into the versatile CRISPR technology, which has been widely applied in site- specific genome editing and has revolutionized biomedical research due to it...Prokaryotic type II adaptive immune systems have been developed into the versatile CRISPR technology, which has been widely applied in site- specific genome editing and has revolutionized biomedical research due to its superior efficiency and flexibility. Recent studies have greatly diversified CRISPR technologies by coupling it with various DNA repair mechanisms and targeting strategies. These new advances have significantly expanded the generation of genetically modified animal models, either by including species in which targeted genetic modification could not be achieved previously, or through introducing complex genetic modifications that take multiple steps and cost years to achieve using traditional methods. Herein, we review the recent developments and applications of CRISPR-based technology in generating various animal models, and discuss the everlasting impact of this new progress on biomedical research.展开更多
基金supported by the Biological BreedingMajor Projects(2023ZD04076)the National Natural Science Foundation of China(32300312)+2 种基金the Innovation Program of Chinses Academy of Agricultural Sciences(CAAS-CSIAF-202303)the Guangdong Basic and Applied Basic Research Foundation(2020B1515120086)the KeyArea Research and Development Program of Guangdong Province(2021B0707010006)。
文摘Rice, a global staple food, is critical for food security. The cultivated Oryza sativa, domesticated from wild O. rufipogon, derives~80%of its 993 identified domestication-related genes from O. rufipogon and 20%from South/Southeast Asian wild O. nivara(Jing et al., 2023). Genes like An-1, BH4, PROG1,SH4, Rc, Rd, and GS3—which regulate awn length, hull color,til er angle, seed shattering, pericarp color, seed length, and thousand-grain weight, respectively—were selected against during domestication to form modern O. sativa(Yu et al., 2021).However, domestication and yield-focused breeding eliminated wild rice's valuable genes(e.g., for disease resistance, stress tolerance, nutrition), narrowing genetic diversity and impeding efforts to meet growing societal demands.
基金supported by the Jilin Science and Technology Development Program,China (20240602032RC)the Jilin Agricultural Science and Technology Innovation Project,China (CXGC2024ZD001)+1 种基金the Jilin Agricultural Science and Technology Innovation Project,China (CXGC2024ZY012)the Jilin Province Development and Reform Commission-Project for Improving the Independent Innovation Capacity of Major Grain Crops,China (2024C002)。
文摘Emerging and powerful genome editing tools,particularly CRISPR/Cas9,are facilitating functional genomics research and accelerating crop improvement(Jiang et al.2021;Cao et al.2023;Chen C et al.2023;Liu et al.2023a).However,the detection and screening of transgenic lines remain major bottlenecks,being time-consuming,labor-intensive,and inefficient during transformation and subsequent mutation identification.A simple and efficient visual marker system plays a critical role in addressing these challenges.Recent studies demonstrated that the GmW1 and RUBY reporter systems were used to obtain visual transgenic soybean(Glycine max) plants(Chen L et al.2023;Chen et al.2024).
基金supported by National Institutes of Health (NIH) Grant R21MH102685the North Shore University Health System 2011 Pilot Award
文摘Schizophrenia（SZ） is a devastating mental disorder afflicting 1% of the population. Recent genome-wide association studies（GWASs） of SZ have identified 〉100 risk loci. However,the causal variants/genes and the causal mechanisms remain largely unknown,which hinders the translation of GWAS fi ndings into disease biology and drug targets. Most risk variants are noncoding,thus likely regulate gene expression. A major mechanism of transcriptional regulation is chromatin remodeling,and open chromatin is a versatile predictor of regulatory sequences. Micro RNA-mediated post-transcriptional regulation plays an important role in SZ pathogenesis. Neurons differentiated from patient-specifi c induced pluripotent stem cells（i PSCs） provide an experimental model to characterize the genetic perturbation of regulatory variants that are often specifi c to cell type and/or developmental stage. The emerging genome-editing technology enables the creation ofisogenic i PSCs and neurons to effi ciently characterize the effects of SZ-associated regulatory variants on SZ-relevant molecular and cellular phenotypes involving dopaminergic,glutamatergic,and GABAergic neurotransmissions. SZ GWAS fi ndings equipped with the emerging functional genomics approaches provide an unprecedented opportunity for understanding new disease biology and identifying novel drug targets.
基金supported by the Agricultural Gongguan Project of Xinjiang Production and Construction Corps,China(NYHXGG,2023AA102)the Innovation Program of Chinese Academy of Agricultural Sciences(CAAS-CSIAF-202402)+1 种基金the Outstanding Youth Foundation of Henan Scientific Committee,China(222300420097)the China Agriculture Research System of MOF and MARA(CARS-15-02).
文摘Genome editing in plants is a powerful strategy that can substantially advance functional genomics research,facilitating the discovery,enhancement,and development of novel traits with significant agricultural implications.Various methodologies,such as zinc finger nucleases(ZFNs),transcription activator-like effector nucleases(TALENs),and CRISPR/Cas systems,have been developed for this purpose.
文摘Precise chromosome engineering has traditionally relied on the Cre-Lox recombination system-an approach in which the enzyme Cre functions like molecular scissors,cutting and rejoining DNA at specific“Lox”sites to add,remove,or flip genomic DNA segments inside living cells.
基金Supported by Scientific Research Start-up Fund for PhD.of Zhaoqing University(210046)the Zhaoqing Science and Technology Innovation Guidance Project(231017174162871,241226220091239).
文摘CRISPR/Cas9 technology, a revolutionary gene-editing tool, has rapidly garnered attention in plant science owing to its simplicity, high editing efficiency, and cost-effectiveness. Besides, it offers unprecedented precision and efficiency in the genetic improvement of fruit trees. To date, this technology has been widely utilized to enhance fruit quality, improve stress resistance, and mediate growth and development. These applications demonstrate its immense potential in fruit tree breeding. Looking ahead, advancements in editing efficiency, expanded application scopes, comprehensive safety assessments, and improved regulatory frameworks are expected to further broaden the role of CRISPR/Cas9 in fruit tree breeding, thereby driving the fruit tree industry toward higher yield, superior quality, enhanced stress resilience, higher efficiency, and contributing to global food security and sustainable agricultural development. This article outlines the fundamental principles of CRISPR/Cas9 gene editing technology, its applications in plants (including fruit trees), and its pivotal role in genetic improvement and germplasm innovation.
基金supported by grants from the National Key Research&Development Plan(Grant Nos.2022YFD1200502,2021YFD1200201)National Natural Science Foundation of China(Grant Nos.32372696,31991182)+6 种基金Wuhan Biological Breeding Major Project(Grant No.2022021302024852)HZAU-AGIS Cooperation Fund(Grant No.SZYJY2023022)Funds for High Quality Development of Hubei Seed Industry(Grant No.HBZY2023B004)Hubei Agriculture Research System(Grant No.2023HBSTX4-06)Hubei Key Research&Development Plan(Grant Nos.2022BBA0066,2022BBA0062)Funds of National Key Laboratory for Germplasm Innovation&Utilization of Horticultural Crops(Grant No.Horti-3Y-2024-008)Key Project of Hubei Hongshan Laboratory(Grant No.2021hszd007).
文摘Tomato,a vegetable of considerable global significance,has been the subject of extensive breeding efforts primarily aimed at achieving high yields and resistance to multiple stressors.However,despite these endeavors,the quality of fruits still cannot fully satisfy the diverse preferences of the majority of consumers.The elucidation of genetic determinants underlying fruit quality traits,coupled with the advancement of gene editing techniques,has significantly contributed to the enhancement of tomato quality.Combining with gene editing technology to improve tomato fruit quality traits represents a viable approach for maximizing the utilization of essential genes in breeding programs.This review provides a comprehensive summary of the significant genes associated with tomato fruit quality traits,as well as an overview of the current advancements and potential avenues for enhancing tomato quality through gene editing technology.Four important aspects of fruit quality-appearance,flavor,nutritional profiles and postharvest properties form the basis of the review,providing a thorough update on the state of research in tomato fruit quality improvement via new gene editing techniques.
文摘Gastric cancer(GC)remains one of the leading causes of cancer-related mortality worldwide,necessitating innovative approaches for its diagnosis and treatment.Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPRassociated protein 9(Cas9),a revolutionary gene-editing technology,has emerged as a powerful tool for unraveling the molecular mechanisms underlying GC and for advancing precision medicine strategies.This review explores the current applications of CRISPR/Cas9 in GC research,including the identification of oncogenes and tumor suppressors,modeling tumor microenvironment interactions,and developing gene-based therapies.We highlight recent breakthroughs in genome editing that have enhanced our understanding of GC pathogenesis and resistance mechanisms to conventional therapies.Additionally,we discuss the potential of CRISPR/Cas9 for therapeutic gene editing in GC,addressing challenges such as off-target effects,delivery methods,and ethical considerations.By summarizing the progress and limitations of CRISPR/Cas9 in GC,this review aims to provide a comprehensive perspective on how this transformative technology could shape future strategies for the prevention,diagnosis,and treatment of GC.
基金funded by the National Research Foundation of Korea(NRF)grant from the Ministry of Science and ICT(MSIT),Republic of Korea(Nos.RS-2024-00407469 and RS-2025-00517964)the BK21 FOUR program of Graduate School,Kyung Hee University(GS-1-JO-NON-20240417)。
文摘Optimizing plant architecture for specific cultivation methods is essential for enhancing fruit productivity.Unlike indeterminate growth plants,the total productivity of determinate growth plants relies on cumulative fruit production and synchronized fruit ripening from both main and axillary shoots.Here,we focused on SlD14and SlMAX1,two key genes involved in the regulation of strigolactone(SL)signaling and biosynthesis,with the goal of maximizing yield and syn chronizing fruit ripening by fine-tuning axillary shoot growth.Using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)technology,we found that the sld14,slmax1,and sld14 slmax1mutant plants exhibited reduced plant height and increased axillary shoot proliferation compared to wild-type plants.However,these mutants showed reduced yield and delayed ripening,likely due to a source-sink imbalance caused by excessive axillary shoot development.A weak sld14 allele displayed a milder phenotype,maintaining total fruit yield and harvest index despite smaller individual fruit size.These findings indicate that allelic variation in SL-related genes can influence plant architecture and yield components.Our results suggest that weak or partial alleles may serve as promising targets for tailoring tomato architecture to space-limited cultivation systems.
基金supported by the Agricultural Science and Technology Innovation Program
文摘Generating mutants bearing multiple gene modifications is essential for determining the functions of gene family members with redundant functions, or for analyzing epistatic re- lationships in genetic pathways. Using conventional methods, mutants with multiple gene mutations are generated by several rounds of intercrossing plants carrying a single mutation and identification of the offspring. This process is both timeconsuming and labor-intensive. Moreover, if the genes of interest are closely linked, multiple mutations can not be generated （Wijnker and de Jong, 2008）.
基金supported by the Strategic Priority Research Program of Chinese Academy of Sciences (No.XDA08040101)the Hundred Talents Program of Chinese Academy of Sciences to Q.Y. Bu+1 种基金the Natural Science Foundation of Heilongjiang (No.ZD2015005)Excellent Academic Leaders of Harbin (No.RC2014XK002003)
文摘Rice is a staple food for more than half of the human population.It has been estimated that by 2030,40%more rice needs to be produced in order to meet the growing demand（Khush,2005）.One of the strategies to improve rice productivity is to enlarge rice growth areas, such as the northward expansion of the growth region in Heilongjiang Province, the northernmost region of China （Li et al., 2015）. However, the northward cultivation is accompanied with daylength extension and temperature decrease, which are unfavor- able for rice, a tropical short-day plant, to complete flowering and seed setting. Thus, the need for early-maturing rice cultivars with extremely low photoperiod sensitivity is urgent.
基金supported in part by Cotton Incorporated and the National Science Foundation(award 1658709)supported by the National Natural Science Foundation of China(No.31700316)+1 种基金the Fundamental Research Funds for the Central Nonprofit Scientific Institution(No.1610172018009)the Natural Science Foundation of Hubei Province(No.2018CFB543),China。
文摘Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010 s,clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)has rapidly been developed into a robust,multifunctional genome editing tool with many uses.Following the discovery of the initial CRISPR/Cas-based system,the technology has been advanced to facilitate a multitude of different functions.These include development as a base editor,prime editor,epigenetic editor,and CRISPR interference(CRISPRi)and CRISPR activator(CRISPRa)gene regulators.It can also be used for chromatin and RNA targeting and imaging.Its applications have proved revolutionary across numerous biological fields,especially in biomedical and agricultural improvement.As a diagnostic tool,CRISPR has been developed to aid the detection and screening of both human and plant diseases,and has even been applied during the current coronavirus disease 2019(COVID-19)pandemic.CRISPR/Cas is also being trialed as a new form of gene therapy for treating various human diseases,including cancers,and has aided drug development.In terms of agricultural breeding,precise targeting of biological pathways via CRISPR/Cas has been key to regulating molecular biosynthesis and allowing modification of proteins,starch,oil,and other functional components for crop improvement.Adding to this,CRISPR/Cas has been shown capable of significantly enhancing both plant tolerance to environmental stresses and overall crop yield via the targeting of various agronomically important gene regulators.Looking to the future,increasing the efficiency and precision of CRISPR/Cas delivery systems and limiting off-target activity are two major challenges for wider application of the technology.This review provides an in-depth overview of current CRISPR development,including the advantages and disadvantages of the technology,recent applications,and future considerations.
基金supported by the National Science Foundation of China(31771808)Ministry of Science and Technology(2015BAD02B0203)+1 种基金National Engineering Laboratory of Crop Molecular Breedingthe Chinese Academy of Agricultural Sciences(Y2017XM03)
文摘Single-guide RNA(sg RNA) is one of the two core components of the CRISPR(clustered regularly interspaced short palindromic repeat)/Cas(CRISPR-associated) genome-editing technology. We established an in vitro Traffic Light Reporter(TLR) system, which is designated as the same colors as traffic lights such as green, red and yellow were produced in cells. The TLR can be readily used in maize mesophyll protoplast for a quick test of promoter activity. The TLR assay indicates the variation in transcription activities of the seven Pol III promoters, from 3.4%(U6-1) to over 21.0%(U6-6). The U6-2 promoter, which was constructed to drive sg RNA expression targeting the Zm Wx1 gene, yielded mutation efficiencies ranging from 48.5% to 97.1%. Based on the reported and unpublished data, the in vitro TLR assay results were confirmed to be a readily system and may be extended to other plant species amenable to efficient genome editing via CRISPR/Cas. Our efforts provide an efficient method of identifying native Pol III-recognized promoters for RNA guide-based genome-editing systems in maize.
基金supported by the National Key Research and Development Program of China(2016YFD0102000)“Breeding of Major New Varieties of Main Grain Crops”Program(2020ABA016)from Department of Science and Technology of Hubei Province.
文摘Grain size is one of the most important factors affecting rice grain quality and yield,and attracts great attention from molecular biologists and breeders.In this study,we engineered a CRISPR/Cas9 system targeting the miR396 recognition site of the rice GS2 gene,which encodes growth-regulating factor 4(OsGRF4)and regulates multiple agronomic traits including grain size,grain quality,nitrogen use efficiency,abiotic stress response,and seed shattering.In contrast to most previous genome editing efforts in which indel mutations were chosen to obtain null mutants,a mutant named GS2^(E) carrying an in-frame 6-bp deletion and 1-bp substitution within the miR396-targeted sequence was identified.GS2^(E) plants showed increased expression of GS2 in consistent with impaired repression by miR396.As expected,the gain-of-function GS2^(E) mutant exhibited multiple beneficial traits including increased grain size and yield and bigger grain length/width ratio.Thousand grain weight and grain yield per plant of GS2^(E) plants were increased by 23.5%and 10.4%,respectively.These improved traits were passed to hybrids in a semidominant way,suggesting that the new GS2^(E) allele has great potential in rice improvement.Taken together,we report new GS2 germplasm and describe a novel gene-editing strategy that can be widely employed to improve grain size and yield in rice.This trait-improvement strategy could be applied to other genes containing miRNA target sites,in particular the conserved miR396-GRF/GIF module that governs plant growth,development and environmental response.
基金This work was supported by the National Key Research and Development Program(Grant No.2018YFD1000200)the National Natural Science Foundation of China(Grant No.31772284).
文摘The quality traits of horticultural crops,including the accumulation of nutrients and flavor substances,morphology,and texture,affect the palatability and nutritional value.For many years,efforts have been made to improve the quality of horticultural crops.The recent establishment of gene editing technology,with its potential applications in horticultural crops,provides a strategy for achieving this goal in a rapid and efficient manner.Here,we summarize research efforts aimed at improving horticultural crop quality through genome editing.We describe specific genome editing systems that have been used and traits that have been targeted in these efforts.Additionally,we discuss limiting factors and future perspectives of genome editing technology in improving horticultural crop qualities in both research and plant breeding.In summary,genome editing technology is emerging as a powerful tool for efficiently and rapidly improving horticultural crop quality,and we believe that the cautious application of genome editing in horticultural crops will generate new germplasms with improved quality in the near future.
基金supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences(Precision Seed Design and Breeding,XDA24020101 and XDA24020310)the National Natural Science Foundation of China(31672015,31788103)the Youth Innovation Promotion Association of the Chinese Academy of Sciences(2020000003)。
文摘MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both academic and commercial use.Here,we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences(YTTN)in plants.Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-Lb Cas12 a system.We develop two variants,MAD7-RR and MAD7-RVR that increase the target range of MAD7,as well as an M-AFID(a MAD7-APOBEC fusion-induced deletion)system that creates predictable deletions from 50-deaminated Cs to the MAD7-cleavage site.Moreover,we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs.Using the CRISPR-MAD7 system,we have obtained regenerated mutant rice and wheat plants with up to 65.6%efficiency.
基金supported by the National Key Technology Research and Development Program of the Ministry of Science and Technology of the People’s Republic of China(No.2015BAI09B03)the National Basic Research Program(973 Program)(No.2013CB35102)+3 种基金the National Natural Science Foundation of China(Nos.31371496 and31301008)the Natural Science Foundation of Hubei Province(No.2013CFB031)Wuhan Youth Chenguang Program of Science and Technology(No.2014072704011259)Tsinghua-Peking Center for Life Sciences
文摘The last couple of years have witnessed an explosion in development of CRISPR-based genome editing technologies in cell lines as well as in model organisms. In this review, we focus on the applications of this popular system in Drosophila. We discuss the effectiveness of the CRISPR/Cas9 systems in terms of delivery, mutagenesis detection, parameters affecting efficiency, and off-target issues, with an emphasis on how to apply this powerful tool to characterize gene functions.
基金supported by a National Transgenic Science and Technology Program (2016ZX08010002)to R.W.a startup fund from the Huazhong Agricultural University
文摘Development of tools for targeted modifications of specific DNA sequences in plants is of great importance to basic plant biology research as well as crop improvement.The ability to cut DNA at specific locations in the genome to generate doublestrand breaks（DSBs）in vivo is a prerequisite for any genome editing efforts.
基金supported by the National Natural Science Foundation of ChinaChina (Grant Nos. 31872051, 32072528)the Foundation of Hubei Hongshan Laboratory (Grant No.2021hszd009)。
文摘Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.
基金supported by funds provided by the Research Grants Council of Hong Kong(CUHK 14104614,TBF16ENG007 and TBF17MED002 to B.F.and 3132966 to W.Y.C.)+1 种基金funds from the Croucher Foundation(CAS16CU01/CAS16401 to W.Y.C.)the National Basic Research Program of China(973 Program,2015CB964700 to Y.L.)
文摘Prokaryotic type II adaptive immune systems have been developed into the versatile CRISPR technology, which has been widely applied in site- specific genome editing and has revolutionized biomedical research due to its superior efficiency and flexibility. Recent studies have greatly diversified CRISPR technologies by coupling it with various DNA repair mechanisms and targeting strategies. These new advances have significantly expanded the generation of genetically modified animal models, either by including species in which targeted genetic modification could not be achieved previously, or through introducing complex genetic modifications that take multiple steps and cost years to achieve using traditional methods. Herein, we review the recent developments and applications of CRISPR-based technology in generating various animal models, and discuss the everlasting impact of this new progress on biomedical research.
