Drug resistance is a major problem faced by tumor cell-targeted drugs.Currently,functional gene screening is the most common strategy for screening drug resistance genes.In recent years,Crispr-cas9 gene editing techno...Drug resistance is a major problem faced by tumor cell-targeted drugs.Currently,functional gene screening is the most common strategy for screening drug resistance genes.In recent years,Crispr-cas9 gene editing technology has been widely used in the functional studies of tumor-related genes due to their characteristics of accuracy,simplicity and efficiency.The principle of CRISPR-Cas9 Library Screening Technology and its application in functional Gene Screening are reviewed.At the same time,the application prospect of the Crispr-Cas9 technology is forecasted.展开更多
OBJECTIVE Cytochrome P450(CYP)3A accounts for nearly 30%of total CYP enzymes in human liver and participates in the metabolism of over 50%of clinical drugs.CYP3A also plays an important role in the chemical metabolism...OBJECTIVE Cytochrome P450(CYP)3A accounts for nearly 30%of total CYP enzymes in human liver and participates in the metabolism of over 50%of clinical drugs.CYP3A also plays an important role in the chemical metabolism,toxicity,and carcinogenicity.New animal models are needed to investigate CYP3A functions.METHODS The CRISPR-Cas9 technology was used to generate Cyp3a1/2 double knockout rat model.The absence of Cyp3a1/2 expression was evaluated through PCR and immunostaining.Metabolic studies of the CYP3A substrates midazolam and nifedipine both in vitro and in vivo were conducted to verify that CYP3A1/2 was functional y inactive in KO rats.In addition,compensatory up-regulation of other P450 genes in Cyp3a1/2 KO rats was detected.RESULTS The Cyp3a1/2 double KO rats were viable and fertile,and had no obvious physiological abnormities.Compared with the wild-type(WT)rat,Cyp3a1/2 expression was completely absent in the liver of the KO rat.In vitro and in vivo metabolic studies of the CYP3A1/2 substrates indicated that CYP3A1/2 was functionally inactive in double KO rats.CONCLUSION The Cyp3a1/2 double KO rat model was successfully generated and characterized.The Cyp3a1/2 KO rats as a novel rodent animal model will be a valuable tool for the study of the physiological and pharmacological roles of CYP3A,and determining whether the absence of CYP3A results in non-CYP mediated metabolism or metabolism by other CYP isoforms.展开更多
Excessive and uncontrollable inflammatory responses in alveoli can dramatically exacerbate pulmonary disease progressions through vigorous cytokine releases,immune cell infiltration and protease-driven tissue damages....Excessive and uncontrollable inflammatory responses in alveoli can dramatically exacerbate pulmonary disease progressions through vigorous cytokine releases,immune cell infiltration and protease-driven tissue damages.It is an urgent need to explore potential drug strategies for mitigating lung inflammation.Protease-activated receptor 2(PAR2)as a vital molecular target principally participates in various inflammatory diseases via intracellular signal transduction.However,it has been rarely reported about the role of PAR2 in lung inflammation.This study applied CRISPR-Cas9 system encoding Cas9 and sg RNA(p Cas9-PAR2)for PAR2 knockout and fabricated an anionic human serum albuminbased nanoparticles to deliver p Cas9-PAR2 with superior inflammation-targeting efficiency and stability(TAP/p Cas9-PAR2).TAP/p Cas9-PAR2 robustly facilitated p Cas9-PAR2 to enter and transfect inflammatory cells,eliciting precise gene editing of PAR2 in vitro and in vivo.Importantly,PAR2 deficiency by TAP/p Cas9-PAR2 effectively and safely promoted macrophage polarization,suppressed proinflammatory cytokine releases and alleviated acute lung inflammation,uncovering a novel value of PAR2.It also revealed that PAR2-mediated pulmonary inflammation prevented by TAP/p Cas9-PAR2was mainly dependent on ERK-mediated NLRP3/IL-1β and NO/i NOS signalling.Therefore,this work indicated PAR2 as a novel target for lung inflammation and provided a potential nanodrug strategy for PAR2 deficiency in treating inflammatory diseases.展开更多
Targeted mutagenesis based on homologous recombination has been a powerful tool for understanding the mechanisms underlying development, normal physiology, and disease. A recent breakthrough in genome engineering tech...Targeted mutagenesis based on homologous recombination has been a powerful tool for understanding the mechanisms underlying development, normal physiology, and disease. A recent breakthrough in genome engineering technology based on the class of RNA-guided endonucleases, such as clustered regularly interspaced short palindromic repeats(CRISPR)-associated Cas9, is further revolutionizing biology and medical studies. The simplicity of the CRISPR-Cas9 system has enabled its widespread applications in generating germline animal models, somatic genome engineering, and functional genomic screening and in treating genetic and infectious diseases. This technology will likely be used in all fields of biomedicine, ranging from basic research to human gene therapy.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
The class 2 clustered regularly interspaced short palindromic repeat (CRISPR) systems have been widely used for simultaneous modification of multiple loci in plants. Traditionally, the type II CRISPR-Cas9 or type V ...The class 2 clustered regularly interspaced short palindromic repeat (CRISPR) systems have been widely used for simultaneous modification of multiple loci in plants. Traditionally, the type II CRISPR-Cas9 or type V CRISPR-Cpfl (also known as Cas12a) system is a two-component transcriptional unit (TCTU) in which the Cas9 or Cpf1 protein is expressed from an RNA polymerase (pol) II promoter, whereas the single guide RNA (sgRNA) is typically expressed from a Pol III promoter, such as U6 or U3 promoter.展开更多
There are an estimated 10000 monogenic diseases affecting tens of millions of individuals worldwide.The application of CRISPR/Cas genome editing tools to treat monogenic diseases is an emerging strategy with the poten...There are an estimated 10000 monogenic diseases affecting tens of millions of individuals worldwide.The application of CRISPR/Cas genome editing tools to treat monogenic diseases is an emerging strategy with the potential to generate personalized treatment approaches for these patients.CRISPR/Cas-based systems are programmable and sequence-specific genome editing tools with the capacity to generate base pair resolution manipulations to DNA or RNA.The complexity of genomic insults resulting in heritable disease requires patientspecific genome editing strategies with consideration of DNA repair pathways,and CRISPR/Cas systems of different types,species,and those with additional enzymatic capacity and/or delivery methods.In this review we aim to discuss broad and multifaceted therapeutic applications of CRISPR/Cas gene editing systems including in harnessing of homology directed repair,non-homologous end joining,microhomology-mediated end joining,and base editing to permanently correct diverse monogenic diseases.展开更多
CRISPR-mediated genome editing is a revolutionary technology for genome manipulation that uses the CRISPR-Cas systems and base editors.Currently,poor efficiency and off-target problems have impeded the application of ...CRISPR-mediated genome editing is a revolutionary technology for genome manipulation that uses the CRISPR-Cas systems and base editors.Currently,poor efficiency and off-target problems have impeded the application of CRISPR systems.The on-target efficiency has been improved in several advanced versions of CRISPR systems,whereas the off-target detection still remains a key challenge.Here,we outline the different versions of CRISPR systems and off-target detection strategies,discuss the merits and limitations of off-target detection methods,and provide potential implications for further gene editing research.展开更多
The 2020 Nobel Prize in Chemistry recognized CRISPR-Cas9,a super-selective and precise gene editing tool.CRISPR-Cas9 has an obvious advantage in editing multiple genes in the same cell,and presents great potential in ...The 2020 Nobel Prize in Chemistry recognized CRISPR-Cas9,a super-selective and precise gene editing tool.CRISPR-Cas9 has an obvious advantage in editing multiple genes in the same cell,and presents great potential in disease treatment and animal model construction.In recent years,CRISPRCas9 has been used to establish a series of rat models of drug metabolism and pharmacokinetics(DMPK),such as Cyp,Abcb1,Oatp1 b2 gene knockout rats.These new rat models are not only widely used in the study of drug metabolism,chemical toxicity,and carcinogenicity,but also promote the study of DMPK related mechanism,and further strengthen the relationship between drug metabolism and pharmacology/toxicology.This review systematically introduces the advantages and disadvantages of CRISPR-Cas9,summarizes the methods of establishing DMPK rat models,discusses the main challenges in this field,and proposes strategies to overcome these problems.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
The CRISPR-Cas system, especially the type II CRISPR-Cas9 system from Streptococcuspyogenes, has rapidly emerged as a popular genome editing tool. The development of Cas9 derivatives further expanded the toolbox of CR...The CRISPR-Cas system, especially the type II CRISPR-Cas9 system from Streptococcuspyogenes, has rapidly emerged as a popular genome editing tool. The development of Cas9 derivatives further expanded the toolbox of CRISPR- Cas9 based genome editing kit. However, therapeutic transla- tion of the CRISPR-Cas9 system in vivo is severely impeded by the absence of an appropriate delivery carrier. The complex- ity and high molecular weight of the CRISPR-Cas9 system, together with the physiological barriers for nucleus targeted cargo transportation have made it a huge challenge for in vivo therapeutic CRISPR-Cas9 delivery. Currently, the main stream carriers for systemic delivery of CRISPR-Cas9 are vi- ral based, such as adeno-associated virus. However, the safety concerns surrounding viral vectors call for the development of non-viral nanocarriers. In this review, we survey the recent advances in the development of non-viral delivery systems for CRISPR-Cas9. Challenges and future directions in this field are also discussed.展开更多
Since its discovery as a bacterial adaptive immune system and its development for genome editing in eukaryotes,the CRISPR technology has revolutionized plant research and precision crop breeding.The CRISPR toolbox hol...Since its discovery as a bacterial adaptive immune system and its development for genome editing in eukaryotes,the CRISPR technology has revolutionized plant research and precision crop breeding.The CRISPR toolbox holds great promise in the production of crops with genetic disease resistance to increase agriculture resilience and reduce chemical crop protection with a strong impact on the environment and public health.In this review,we provide an extensive overviewon recent breakthroughs in CRISPR technology,including the newly developed prime editing system that allows precision gene editing in plants.We present how each CRISPR tool can be selected for optimal use in accordance with its specific strengths and limitations,and illustrate how the CRISPR toolbox can foster the development of genetically pathogen-resistant crops for sustainable agriculture.展开更多
基金Science and technology project of Liuzhou(No.2019BJ10612)。
文摘Drug resistance is a major problem faced by tumor cell-targeted drugs.Currently,functional gene screening is the most common strategy for screening drug resistance genes.In recent years,Crispr-cas9 gene editing technology has been widely used in the functional studies of tumor-related genes due to their characteristics of accuracy,simplicity and efficiency.The principle of CRISPR-Cas9 Library Screening Technology and its application in functional Gene Screening are reviewed.At the same time,the application prospect of the Crispr-Cas9 technology is forecasted.
基金The project supported by National Natural Science Foundation of China(81301908)the Science and Technology Commission of Shanghai Municipality(15140904700,13ZR1412600,14DZ2270100)
文摘OBJECTIVE Cytochrome P450(CYP)3A accounts for nearly 30%of total CYP enzymes in human liver and participates in the metabolism of over 50%of clinical drugs.CYP3A also plays an important role in the chemical metabolism,toxicity,and carcinogenicity.New animal models are needed to investigate CYP3A functions.METHODS The CRISPR-Cas9 technology was used to generate Cyp3a1/2 double knockout rat model.The absence of Cyp3a1/2 expression was evaluated through PCR and immunostaining.Metabolic studies of the CYP3A substrates midazolam and nifedipine both in vitro and in vivo were conducted to verify that CYP3A1/2 was functional y inactive in KO rats.In addition,compensatory up-regulation of other P450 genes in Cyp3a1/2 KO rats was detected.RESULTS The Cyp3a1/2 double KO rats were viable and fertile,and had no obvious physiological abnormities.Compared with the wild-type(WT)rat,Cyp3a1/2 expression was completely absent in the liver of the KO rat.In vitro and in vivo metabolic studies of the CYP3A1/2 substrates indicated that CYP3A1/2 was functionally inactive in double KO rats.CONCLUSION The Cyp3a1/2 double KO rat model was successfully generated and characterized.The Cyp3a1/2 KO rats as a novel rodent animal model will be a valuable tool for the study of the physiological and pharmacological roles of CYP3A,and determining whether the absence of CYP3A results in non-CYP mediated metabolism or metabolism by other CYP isoforms.
基金supported by the National Natural Science Foundation of China(Nos.82003784 and 81872789)the Fundamental Research Funds for the Central Universities(No.2682022ZTPY037,China)Large Instruments Open Foundation of Southwest Jiaotong University(No.2022SRII-046,China)。
文摘Excessive and uncontrollable inflammatory responses in alveoli can dramatically exacerbate pulmonary disease progressions through vigorous cytokine releases,immune cell infiltration and protease-driven tissue damages.It is an urgent need to explore potential drug strategies for mitigating lung inflammation.Protease-activated receptor 2(PAR2)as a vital molecular target principally participates in various inflammatory diseases via intracellular signal transduction.However,it has been rarely reported about the role of PAR2 in lung inflammation.This study applied CRISPR-Cas9 system encoding Cas9 and sg RNA(p Cas9-PAR2)for PAR2 knockout and fabricated an anionic human serum albuminbased nanoparticles to deliver p Cas9-PAR2 with superior inflammation-targeting efficiency and stability(TAP/p Cas9-PAR2).TAP/p Cas9-PAR2 robustly facilitated p Cas9-PAR2 to enter and transfect inflammatory cells,eliciting precise gene editing of PAR2 in vitro and in vivo.Importantly,PAR2 deficiency by TAP/p Cas9-PAR2 effectively and safely promoted macrophage polarization,suppressed proinflammatory cytokine releases and alleviated acute lung inflammation,uncovering a novel value of PAR2.It also revealed that PAR2-mediated pulmonary inflammation prevented by TAP/p Cas9-PAR2was mainly dependent on ERK-mediated NLRP3/IL-1β and NO/i NOS signalling.Therefore,this work indicated PAR2 as a novel target for lung inflammation and provided a potential nanodrug strategy for PAR2 deficiency in treating inflammatory diseases.
基金supported by the Chinese National Key Program on Basic Research (2012CB945103, 2011CB504202)National Natural Science Foundation of China (31430057)
文摘Targeted mutagenesis based on homologous recombination has been a powerful tool for understanding the mechanisms underlying development, normal physiology, and disease. A recent breakthrough in genome engineering technology based on the class of RNA-guided endonucleases, such as clustered regularly interspaced short palindromic repeats(CRISPR)-associated Cas9, is further revolutionizing biology and medical studies. The simplicity of the CRISPR-Cas9 system has enabled its widespread applications in generating germline animal models, somatic genome engineering, and functional genomic screening and in treating genetic and infectious diseases. This technology will likely be used in all fields of biomedicine, ranging from basic research to human gene therapy.
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.
文摘The class 2 clustered regularly interspaced short palindromic repeat (CRISPR) systems have been widely used for simultaneous modification of multiple loci in plants. Traditionally, the type II CRISPR-Cas9 or type V CRISPR-Cpfl (also known as Cas12a) system is a two-component transcriptional unit (TCTU) in which the Cas9 or Cpf1 protein is expressed from an RNA polymerase (pol) II promoter, whereas the single guide RNA (sgRNA) is typically expressed from a Pol III promoter, such as U6 or U3 promoter.
文摘There are an estimated 10000 monogenic diseases affecting tens of millions of individuals worldwide.The application of CRISPR/Cas genome editing tools to treat monogenic diseases is an emerging strategy with the potential to generate personalized treatment approaches for these patients.CRISPR/Cas-based systems are programmable and sequence-specific genome editing tools with the capacity to generate base pair resolution manipulations to DNA or RNA.The complexity of genomic insults resulting in heritable disease requires patientspecific genome editing strategies with consideration of DNA repair pathways,and CRISPR/Cas systems of different types,species,and those with additional enzymatic capacity and/or delivery methods.In this review we aim to discuss broad and multifaceted therapeutic applications of CRISPR/Cas gene editing systems including in harnessing of homology directed repair,non-homologous end joining,microhomology-mediated end joining,and base editing to permanently correct diverse monogenic diseases.
基金supported by the grants 81771230(W.C.),31922048(E.Z.)and 31522037(H.Y.)from the National Natural Science Foundation of China.
文摘CRISPR-mediated genome editing is a revolutionary technology for genome manipulation that uses the CRISPR-Cas systems and base editors.Currently,poor efficiency and off-target problems have impeded the application of CRISPR systems.The on-target efficiency has been improved in several advanced versions of CRISPR systems,whereas the off-target detection still remains a key challenge.Here,we outline the different versions of CRISPR systems and off-target detection strategies,discuss the merits and limitations of off-target detection methods,and provide potential implications for further gene editing research.
基金supported in part by grants from the National Natural Science Foundation of China(No.81773808)the Science and Technology Commission of Shanghai Municipality(Nos.17140901000,17140901001 and 18430760400,China)。
文摘The 2020 Nobel Prize in Chemistry recognized CRISPR-Cas9,a super-selective and precise gene editing tool.CRISPR-Cas9 has an obvious advantage in editing multiple genes in the same cell,and presents great potential in disease treatment and animal model construction.In recent years,CRISPRCas9 has been used to establish a series of rat models of drug metabolism and pharmacokinetics(DMPK),such as Cyp,Abcb1,Oatp1 b2 gene knockout rats.These new rat models are not only widely used in the study of drug metabolism,chemical toxicity,and carcinogenicity,but also promote the study of DMPK related mechanism,and further strengthen the relationship between drug metabolism and pharmacology/toxicology.This review systematically introduces the advantages and disadvantages of CRISPR-Cas9,summarizes the methods of establishing DMPK rat models,discusses the main challenges in this field,and proposes strategies to overcome these problems.
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 the grants from North Carolina TraCS, NIH’s Clinical and Translational Science Awards (CTSA, 1UL1TR001111) at UNC-CHSloan Fellowship Award from the Alfred P. Sloan Foundation
文摘The CRISPR-Cas system, especially the type II CRISPR-Cas9 system from Streptococcuspyogenes, has rapidly emerged as a popular genome editing tool. The development of Cas9 derivatives further expanded the toolbox of CRISPR- Cas9 based genome editing kit. However, therapeutic transla- tion of the CRISPR-Cas9 system in vivo is severely impeded by the absence of an appropriate delivery carrier. The complex- ity and high molecular weight of the CRISPR-Cas9 system, together with the physiological barriers for nucleus targeted cargo transportation have made it a huge challenge for in vivo therapeutic CRISPR-Cas9 delivery. Currently, the main stream carriers for systemic delivery of CRISPR-Cas9 are vi- ral based, such as adeno-associated virus. However, the safety concerns surrounding viral vectors call for the development of non-viral nanocarriers. In this review, we survey the recent advances in the development of non-viral delivery systems for CRISPR-Cas9. Challenges and future directions in this field are also discussed.
基金supported by the Investissement d’Avenir program of the French National Agency of Research for the project GENIUS(ANR-11-BTBR-0001_GENIUS)the Institut Carnot Plant2Pro program for the project POTATOCRISPsupported by the ANR project Immunereceptor(ANR-15-CE20-0007).
文摘Since its discovery as a bacterial adaptive immune system and its development for genome editing in eukaryotes,the CRISPR technology has revolutionized plant research and precision crop breeding.The CRISPR toolbox holds great promise in the production of crops with genetic disease resistance to increase agriculture resilience and reduce chemical crop protection with a strong impact on the environment and public health.In this review,we provide an extensive overviewon recent breakthroughs in CRISPR technology,including the newly developed prime editing system that allows precision gene editing in plants.We present how each CRISPR tool can be selected for optimal use in accordance with its specific strengths and limitations,and illustrate how the CRISPR toolbox can foster the development of genetically pathogen-resistant crops for sustainable agriculture.