Dear Editor,Despite tremendous effort devoted to the development of antiretroviral therapies to combat HIV over the past decades, AIDS remains one of the most important global infectious diseases. According to UNAIDS ...Dear Editor,Despite tremendous effort devoted to the development of antiretroviral therapies to combat HIV over the past decades, AIDS remains one of the most important global infectious diseases. According to UNAIDS report on the global AIDS epidemic in 2016, the estimated number of people living with HIV rose from 7.5 million in 2010 to 36.7 million in 2015. Furthermore, drug-resistance HIV strains have recently been reported （Wensing et al., 2017）. Therefore, it is important to develop new therapies to eliminate HIV in the patients. Immortalized cell lines representing the major targets of HIV in human are important for HIV research and therapeutic development.展开更多
Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated 9(CRISPR/Cas9)system has recently become one popular technology due to its efficiency,precision,and simplicity compared with other genome edi...Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated 9(CRISPR/Cas9)system has recently become one popular technology due to its efficiency,precision,and simplicity compared with other genome editing tools such as Zinc Finger Nucleases(ZFNs)and Transcription Activator Like Effector Nucleases(TALENs).Horticultural crops provide energy and health-keeping nutrients to humankind.Genome-editing technology has become widely adopted in horticultural breeding with the increasing demand for high yield and better-quality horticultural crops.Here,we describe the CRISPR/Cas9 system construction,its optimization,including sgRNA promoter,sgRNA design,Cas9 protein promoter,SpCas9 variants and orthologs,and vector delivery methods.We also summarized the application of this technology in horticultural plants for stress responses enhancement,fruit quality improvement,and cultivation traits modification.This detailed review was compiled to help establish comprehensive understanding of the CRISPR/Cas9 systems and provide a reference for further developing this technology to manipulate horticultural plant traits effectively.展开更多
Yarrowia lipolytica is a promising host for producing valuable chemicals owing to its robustness and metabolic versatility.Efficient genome editing tools are essential for advancing its biotechnological applications.A...Yarrowia lipolytica is a promising host for producing valuable chemicals owing to its robustness and metabolic versatility.Efficient genome editing tools are essential for advancing its biotechnological applications.Although CRISPR/Cas9 technology has been applied in Y.lipolytica,achieving a consistently high editing performance re-mains challenging owing to the low homologous recombination efficiency and variability in system components.In this study,we optimized CRISPR/Cas9-mediated genome editing in Y.lipolytica to enhance its editing effi-ciency.Using the RNA polymerase III promoter SCR1-tRNA for sgRNA expression,we achieved a gene disruption efficiency of 92.5%.The tRNA-sgRNA architecture enabled a dual gene disruption efficiency of 57.5%.KU70 deletion in the Cas9 system increased the integration efficiency to 92.5%,and Rad52 and Sae2 overexpression boosted homologous recombination.The introduction of Cas9D147Y,P411T(iCas9)enhanced the efficiency of both gene disruption and genome integration.This study provides a powerful tool for efficient gene editing in Y.lipolytica,which will accelerate the construction of yeast cell factories.展开更多
The recently developed RNA-guided clustered regularly interspaced short palindromic repeat （CRISPR）/CRISPR-associated 9 （Cas9） nuclease system has progressed to be an invaluable technology for genome manipulation ...The recently developed RNA-guided clustered regularly interspaced short palindromic repeat （CRISPR）/CRISPR-associated 9 （Cas9） nuclease system has progressed to be an invaluable technology for genome manipulation in somatic cell types and germline model organisms. While the unprecedented advance in human embryo gene editing research has great potential in next-generation therapeutics, it raises various ethical concerns that need to be addressed before being translated for clinical use. Here, we discuss the current and potential applications of CRISPR/Cas9 technology and its limitations in clinical applications, as well as ethical and legal considerations in the treatment, disease prevention or disability in somatic cells or human embryo via gene editing.展开更多
Comprehensive Summary Currently,CRISPR/Cas9 technology has found widespread applications across various domains.However,the utility of CRISPR/Cas9 is encumbered by issues pertaining to its reliability and safety,prima...Comprehensive Summary Currently,CRISPR/Cas9 technology has found widespread applications across various domains.However,the utility of CRISPR/Cas9 is encumbered by issues pertaining to its reliability and safety,primarily stemming from the uncontrolled activity of the system.Therefore,the design and development of CRISPR/Cas9 systems with controllable activity is of paramount importance.Biotin,characterized by its small molecular weight,and streptavidin,distinguished by its substantial spatial steric hindrance,can be harnessed as an ideal OFF switch(termed a"bioactivity brake")due to their interaction characteristics.In this work,we present a strategy that employs the streptavidin-biotin interaction as a"brake system"for CRISPR/Cas9,effectively allowing for the shutdown of the enzymatic activity of CRISPR/Cas9.展开更多
目的探究盘状结构域受体1(discoidin domain receptors,DDR1)在慢性结肠炎小鼠肠道炎症及肠纤维化中的功能。方法针对DDR1基因exon4设计并合成gRNA序列,与编码Cas9的mRNA混合显微注射入C57BL/6小鼠受精卵内,构建DDR1基因突变小鼠。选择F...目的探究盘状结构域受体1(discoidin domain receptors,DDR1)在慢性结肠炎小鼠肠道炎症及肠纤维化中的功能。方法针对DDR1基因exon4设计并合成gRNA序列,与编码Cas9的mRNA混合显微注射入C57BL/6小鼠受精卵内,构建DDR1基因突变小鼠。选择F4代基因敲除纯合子小鼠(DDR1^(-/-))和野生型C57BL/6小鼠,通过葡聚糖硫酸钠(DSS)诱导慢性结肠炎模型。比较两组小鼠疾病活动指数(DAI)、体质量、结肠长度及组织病理学变化。ELISA法检测外周血细胞因子的含量,Western blot法检测小鼠结肠纤维化相关蛋白表达。结果PCR法检测结果显示利用CRISPR/Cas9技术成功构建DDR1^(-/-)小鼠;循环饮用DSS后,与野生型小鼠相比,DDR1^(-/-)小鼠体质量下降减缓,DAI评分及肠组织病理学评分降低,结肠短缩减轻(5.81±0.11 cm vs 5.19±0.05 cm,P<0.05);血清IL-1β、TNF-α及TGF-β水平下调(P<0.01);Masson染色胶原容积评分显示肠纤维化减轻(DSS-WT vs DSS-DDR1,41.43±0.08 cm vs 21.37±0.07 cm,P<0.01);肠组织TGF-β、α-SMA和COL1A1的蛋白表达下降。结论DDR1参与慢性结肠炎的发生,敲除DDR1基因可减轻慢性结肠炎肠道炎症及纤维化的症状和疾病进程。展开更多
Pyricularia oryzae anamorph of Magnaporthe oryzae is one of the most notorious fungal pathogens causing severe economic loss in rice production worldwide. Various methods, viz. cultural, biological and molecular appro...Pyricularia oryzae anamorph of Magnaporthe oryzae is one of the most notorious fungal pathogens causing severe economic loss in rice production worldwide. Various methods, viz. cultural, biological and molecular approaches, are utilized to counteract this pathogen. Moreover, some tolerant or resistant rice varieties have been developed with the help of breeding programmes. Isolation and molecular characterization of different blast resistance genes now open the gate for new possibilities to elucidate the actual allelic variants of these genes via various molecular breeding and transgenic approaches. However, the behavioral pattern of this fungus breakups the resistance barriers in the resistant or tolerant rice varieties. This host-pathogen barrier will be possibly countered in future research by comparative genomics data from available genome sequence data of rice and M. oryzae for durable resistance. Present review emphasized fascinating recent updates, new molecular breeding approaches, transgenic and genomics approaches(i.e. mi RNA and genome editing) for the management of blast disease in rice. The updated information will be helpful for the durable, resistance breeding programme in rice against blast pathogen.展开更多
The emerging pests and phytopathogens have reduced the crop yield and quality, which hasthreatened the global food security. Traditional breeding methods, molecular marker-based breedingapproaches and use of genetical...The emerging pests and phytopathogens have reduced the crop yield and quality, which hasthreatened the global food security. Traditional breeding methods, molecular marker-based breedingapproaches and use of genetically modified crops have played a crucial role in strengthening the foodsecurity worldwide. However, their usages in crop improvement have been highly limited due to multiplecaveats. Genome editing tools like transcriptional activator-like effector nucleases and clustered regularlyinterspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 (CRISPR/Cas9) haveeffectively overcome limitations of the conventional breeding methods and are being widely accepted forimprovement of crops. Among the genome editing tools, the CRISPR/Cas9 system has emerged as themost powerful tool of genome editing because of its efficiency, amicability, flexibility, low cost andadaptability. Accumulated evidences indicate that genome editing has great potential in improving thedisease resistance in crop plants. In this review, we offered a brief introduction to the mechanisms of differentgenome editing systems and then discussed recent developments in CRISPR/Cas9 system-based genomeediting towards enhancement of rice disease resistance by different strategies. This review also discussed thepossible applications of recently developed genome editing approaches like CRISPR/Cas12a (formerlyknown as Cpf1) and base editors for enhancement of rice disease resistance.展开更多
Soil salinization has emerged as a major threat affecting crop yields.Global warming leads to a massive loss of terrestrial water and makes soils saltier.Cultivating salt-tolerant crops is the major strategy adopted f...Soil salinization has emerged as a major threat affecting crop yields.Global warming leads to a massive loss of terrestrial water and makes soils saltier.Cultivating salt-tolerant crops is the major strategy adopted for utilizing these salinized soils.Sea Rice 86(SR86)is one such elite salt-tolerant rice variety derived from ancient indica rice.However,SR86 has multiple wild traits,such as tallness and strong photoperiod sensitivity(PS),which have limited its application in agricultural production.In this study,we edited 13 genes responsible for 10 traits in SR86 to develop an improved SR86M line by using clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9 multiplex-genome-editing technology,high-throughput sequencing,crossing,and progeny selection.Subsequent analysis of SR86M detected nine genes with expected mutations,leading to changes in seven traits,including improvements of plant architecture,plant height and PS decreased,grain number,grain length,fragrance,and nitrogen utilization efficiency increased.The improved agronomic traits in SR86M are similar to modern cultivated rice,along with elite salt tolerance like SR86,indicating suitability for potential cultivation.Our results also reveal the efficiency of multiplex-genome-editing in directional improvement of crop varieties.展开更多
文摘Dear Editor,Despite tremendous effort devoted to the development of antiretroviral therapies to combat HIV over the past decades, AIDS remains one of the most important global infectious diseases. According to UNAIDS report on the global AIDS epidemic in 2016, the estimated number of people living with HIV rose from 7.5 million in 2010 to 36.7 million in 2015. Furthermore, drug-resistance HIV strains have recently been reported （Wensing et al., 2017）. Therefore, it is important to develop new therapies to eliminate HIV in the patients. Immortalized cell lines representing the major targets of HIV in human are important for HIV research and therapeutic development.
基金supported by grants from the National Key R&D Program of China (Grant No. 2018YFD1000100)National Natural Science Foundation of China (Grant No. 31972378)Agricultural Variety Improvement Project of Shandong Province (Grant No. 2019LZGC007)
文摘Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated 9(CRISPR/Cas9)system has recently become one popular technology due to its efficiency,precision,and simplicity compared with other genome editing tools such as Zinc Finger Nucleases(ZFNs)and Transcription Activator Like Effector Nucleases(TALENs).Horticultural crops provide energy and health-keeping nutrients to humankind.Genome-editing technology has become widely adopted in horticultural breeding with the increasing demand for high yield and better-quality horticultural crops.Here,we describe the CRISPR/Cas9 system construction,its optimization,including sgRNA promoter,sgRNA design,Cas9 protein promoter,SpCas9 variants and orthologs,and vector delivery methods.We also summarized the application of this technology in horticultural plants for stress responses enhancement,fruit quality improvement,and cultivation traits modification.This detailed review was compiled to help establish comprehensive understanding of the CRISPR/Cas9 systems and provide a reference for further developing this technology to manipulate horticultural plant traits effectively.
基金supported by the National Natural Science Founda-tion of China(U23A20268)the National Natural Science Foundation of Shandong Province(ZR2022ZD24)the Taishan Scholar Project of Shandong Province(tsqn202312061).
文摘Yarrowia lipolytica is a promising host for producing valuable chemicals owing to its robustness and metabolic versatility.Efficient genome editing tools are essential for advancing its biotechnological applications.Although CRISPR/Cas9 technology has been applied in Y.lipolytica,achieving a consistently high editing performance re-mains challenging owing to the low homologous recombination efficiency and variability in system components.In this study,we optimized CRISPR/Cas9-mediated genome editing in Y.lipolytica to enhance its editing effi-ciency.Using the RNA polymerase III promoter SCR1-tRNA for sgRNA expression,we achieved a gene disruption efficiency of 92.5%.The tRNA-sgRNA architecture enabled a dual gene disruption efficiency of 57.5%.KU70 deletion in the Cas9 system increased the integration efficiency to 92.5%,and Rad52 and Sae2 overexpression boosted homologous recombination.The introduction of Cas9D147Y,P411T(iCas9)enhanced the efficiency of both gene disruption and genome integration.This study provides a powerful tool for efficient gene editing in Y.lipolytica,which will accelerate the construction of yeast cell factories.
文摘The recently developed RNA-guided clustered regularly interspaced short palindromic repeat （CRISPR）/CRISPR-associated 9 （Cas9） nuclease system has progressed to be an invaluable technology for genome manipulation in somatic cell types and germline model organisms. While the unprecedented advance in human embryo gene editing research has great potential in next-generation therapeutics, it raises various ethical concerns that need to be addressed before being translated for clinical use. Here, we discuss the current and potential applications of CRISPR/Cas9 technology and its limitations in clinical applications, as well as ethical and legal considerations in the treatment, disease prevention or disability in somatic cells or human embryo via gene editing.
基金the National Natural Science Foundation of China(Nos.22177089,21721005,92153303,22037004,22177088)the Fundamental Research Funds for the Central Universities(2042023kf0204)Translational Medicine and Interdisciplinary Research Joint Fund of Zhongnan Hospital of Wuhan University(Grant No.ZNJC202309).
文摘Comprehensive Summary Currently,CRISPR/Cas9 technology has found widespread applications across various domains.However,the utility of CRISPR/Cas9 is encumbered by issues pertaining to its reliability and safety,primarily stemming from the uncontrolled activity of the system.Therefore,the design and development of CRISPR/Cas9 systems with controllable activity is of paramount importance.Biotin,characterized by its small molecular weight,and streptavidin,distinguished by its substantial spatial steric hindrance,can be harnessed as an ideal OFF switch(termed a"bioactivity brake")due to their interaction characteristics.In this work,we present a strategy that employs the streptavidin-biotin interaction as a"brake system"for CRISPR/Cas9,effectively allowing for the shutdown of the enzymatic activity of CRISPR/Cas9.
文摘目的探究盘状结构域受体1(discoidin domain receptors,DDR1)在慢性结肠炎小鼠肠道炎症及肠纤维化中的功能。方法针对DDR1基因exon4设计并合成gRNA序列,与编码Cas9的mRNA混合显微注射入C57BL/6小鼠受精卵内,构建DDR1基因突变小鼠。选择F4代基因敲除纯合子小鼠(DDR1^(-/-))和野生型C57BL/6小鼠,通过葡聚糖硫酸钠(DSS)诱导慢性结肠炎模型。比较两组小鼠疾病活动指数(DAI)、体质量、结肠长度及组织病理学变化。ELISA法检测外周血细胞因子的含量,Western blot法检测小鼠结肠纤维化相关蛋白表达。结果PCR法检测结果显示利用CRISPR/Cas9技术成功构建DDR1^(-/-)小鼠;循环饮用DSS后,与野生型小鼠相比,DDR1^(-/-)小鼠体质量下降减缓,DAI评分及肠组织病理学评分降低,结肠短缩减轻(5.81±0.11 cm vs 5.19±0.05 cm,P<0.05);血清IL-1β、TNF-α及TGF-β水平下调(P<0.01);Masson染色胶原容积评分显示肠纤维化减轻(DSS-WT vs DSS-DDR1,41.43±0.08 cm vs 21.37±0.07 cm,P<0.01);肠组织TGF-β、α-SMA和COL1A1的蛋白表达下降。结论DDR1参与慢性结肠炎的发生,敲除DDR1基因可减轻慢性结肠炎肠道炎症及纤维化的症状和疾病进程。
文摘Pyricularia oryzae anamorph of Magnaporthe oryzae is one of the most notorious fungal pathogens causing severe economic loss in rice production worldwide. Various methods, viz. cultural, biological and molecular approaches, are utilized to counteract this pathogen. Moreover, some tolerant or resistant rice varieties have been developed with the help of breeding programmes. Isolation and molecular characterization of different blast resistance genes now open the gate for new possibilities to elucidate the actual allelic variants of these genes via various molecular breeding and transgenic approaches. However, the behavioral pattern of this fungus breakups the resistance barriers in the resistant or tolerant rice varieties. This host-pathogen barrier will be possibly countered in future research by comparative genomics data from available genome sequence data of rice and M. oryzae for durable resistance. Present review emphasized fascinating recent updates, new molecular breeding approaches, transgenic and genomics approaches(i.e. mi RNA and genome editing) for the management of blast disease in rice. The updated information will be helpful for the durable, resistance breeding programme in rice against blast pathogen.
基金the China Priority Program-Breeding of Seven Major Crops(Grant No.2017YFD01100100)the Innovation Program of Chinese Academy of Agricultural Sciences(Grant No.01-ICS)the Talented Young Scientist Program of China(Grant No.India-17-01).
文摘The emerging pests and phytopathogens have reduced the crop yield and quality, which hasthreatened the global food security. Traditional breeding methods, molecular marker-based breedingapproaches and use of genetically modified crops have played a crucial role in strengthening the foodsecurity worldwide. However, their usages in crop improvement have been highly limited due to multiplecaveats. Genome editing tools like transcriptional activator-like effector nucleases and clustered regularlyinterspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 (CRISPR/Cas9) haveeffectively overcome limitations of the conventional breeding methods and are being widely accepted forimprovement of crops. Among the genome editing tools, the CRISPR/Cas9 system has emerged as themost powerful tool of genome editing because of its efficiency, amicability, flexibility, low cost andadaptability. Accumulated evidences indicate that genome editing has great potential in improving thedisease resistance in crop plants. In this review, we offered a brief introduction to the mechanisms of differentgenome editing systems and then discussed recent developments in CRISPR/Cas9 system-based genomeediting towards enhancement of rice disease resistance by different strategies. This review also discussed thepossible applications of recently developed genome editing approaches like CRISPR/Cas12a (formerlyknown as Cpf1) and base editors for enhancement of rice disease resistance.
基金funded by grants from the National Key R&D Program of China(2023ZD04073)the Major Program of Guangdong Basic and Applied Research(2019B030302006)+1 种基金the open competition program of top 10 critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province(2022SDZG05)Guangdong Basic Research Center of Excellence for Precise Breeding of Future Crops Major Project(FCBRCE-202502,FCBRCE-202504).
文摘Soil salinization has emerged as a major threat affecting crop yields.Global warming leads to a massive loss of terrestrial water and makes soils saltier.Cultivating salt-tolerant crops is the major strategy adopted for utilizing these salinized soils.Sea Rice 86(SR86)is one such elite salt-tolerant rice variety derived from ancient indica rice.However,SR86 has multiple wild traits,such as tallness and strong photoperiod sensitivity(PS),which have limited its application in agricultural production.In this study,we edited 13 genes responsible for 10 traits in SR86 to develop an improved SR86M line by using clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9 multiplex-genome-editing technology,high-throughput sequencing,crossing,and progeny selection.Subsequent analysis of SR86M detected nine genes with expected mutations,leading to changes in seven traits,including improvements of plant architecture,plant height and PS decreased,grain number,grain length,fragrance,and nitrogen utilization efficiency increased.The improved agronomic traits in SR86M are similar to modern cultivated rice,along with elite salt tolerance like SR86,indicating suitability for potential cultivation.Our results also reveal the efficiency of multiplex-genome-editing in directional improvement of crop varieties.
