Soybean mosaic virus(SMV),an RNA virus,is the most common and destructive pathogenic virus in soybean fields.The newly developed CRISPR/Cas immune system has provided a novel strategy for improving plant resistance to...Soybean mosaic virus(SMV),an RNA virus,is the most common and destructive pathogenic virus in soybean fields.The newly developed CRISPR/Cas immune system has provided a novel strategy for improving plant resistance to viruses;hence,this study aimed to engineer SMV resistance in soybean using this system.Specifically,multiple sgRNAs were designed to target positive-and/or negative-sense strands of the SMV HC-Pro gene.Subsequently,the corresponding CRISPR/CasRx vectors were constructed and transformed into soybeans.After inoculation with SMV,39.02%,35.77%,and 18.70%of T_(1)plants were confirmed to be highly resistant(HR),resistant(R),and mildly resistant(MR)to SMV,respectively,whereas only 6.50%were identified as susceptible(S).Additionally,qRT-PCR and DAS-ELISA showed that,both at 15 and 30 d post-inoculation(dpi),SMV accumulation significantly decreased or was even undetectable in HR and R plants,followed by MR and S plants.Additionally,the expression level of the CasRx gene varied in almost all T_(1)plants with different resistance level,both at 15 and 30 dpi.Furthermore,when SMV resistance was evaluated in the T_(2)generation,the results were similar to those recorded for the T_(1)generation.These findings provide new insights into the application of the CRISPR/CasRx system for soybean improvement and offer a promising alternative strategy for breeding for resistance to biotic stress that will contribute to the development of SMV-immune soybean germplasm to accelerate progress towards greater soybean crop productivity.展开更多
Osteoarthritis(OA)is a chronic degenerative joint disease.Currently,OA is incur-able.Abnormal activation of canonical Wnt/b-catenin or Indian hedgehog(Ihh)signaling could lead to OA development and progression.This st...Osteoarthritis(OA)is a chronic degenerative joint disease.Currently,OA is incur-able.Abnormal activation of canonical Wnt/b-catenin or Indian hedgehog(Ihh)signaling could lead to OA development and progression.This study aimed to determine if targeting b-catenin and Ihh signaling could yield an effective therapeutic intervention for OA disease.CRISPR/CasRx is a new RNA interference tool that can precisely and efficiently cleave single-strand RNAs.In this study,we screened CRISPR-derived RNA(crRNA)targeting Ctnnb1 and Smo in vitro and selected two optimal crRNAs for each gene.CasRx-mediated Ctnnb1 and Smo knockdown showed high efficiency and specificity with no obvious off-target effects in vitro.We then per-formed intra-articular injection of selected crRNAs driven by the adeno-associated virus into an OA mouse model.Micro-CT,histological,and histomorphometric analyses were conducted to evaluate the efficacy of CasRx approach on OA treatment.We found that the knockdown of Ctnnb1 and Smo decelerated pathological damage in the keen joint of the experimental OA mouse model.Our findings suggest that CasRx-mediated Ctnnb1 and Smo knockdown could be a potential strategy for OA treatment.展开更多
The RNA editing tool CRISPR-CasRx has provided a platform for a range of transcriptome analysis tools and therapeutic approaches with its broad efficacy and high specificity.To enable the application of CasRx in vivo,...The RNA editing tool CRISPR-CasRx has provided a platform for a range of transcriptome analysis tools and therapeutic approaches with its broad efficacy and high specificity.To enable the application of CasRx in vivo,we established a Credependent CasRx knock-in mouse.Using these mice,we specifically knocked down the expression of Meis1 and Hoxb13 in cardiomyocytes,which induced cardiac regeneration after myocardial infarction.We also knocked down the lnc RNA Mhrt in cardiomyocytes with the CasRx knock-in mice,causing hypertrophic cardiomyopathy.In summary,we generated a Credependent CasRx knock-in mouse that can efficiently knock down coding gene and lnc RNA expression in specific somatic cells.This in vivo CRISPR-CasRx system is promising for gene function research and disease modeling.展开更多
The CRISPR-Cas13 system,an RNA-guided editing tool,has emerged as a highly efficient and stable RNA editing technique.Although the CRISPR-Cas13 system has been developed in several insect species,its application in le...The CRISPR-Cas13 system,an RNA-guided editing tool,has emerged as a highly efficient and stable RNA editing technique.Although the CRISPR-Cas13 system has been developed in several insect species,its application in lepidopterans has not yet been reported.In the present study,we evaluated the RNA cleavage activity of the CRISPR-Cas13 system in the silkworm(Bombyx mori),a model lepidopteran insect,both ex vivo and in vivo.We established two stable silkworm BmE cell lines expressing PspCas13b and CasRx,respectively.Further analysis demonstrated that both PspCas13b and CasRx effectively down-regulated the transcription of exogenouslyintroduced target and endogenous genes in these cell lines.In addition,we generated two transgenic silkworm strains,one expressing CasRx and the other expressing RNA-guided CRISPR RNA targeting Sex combs reduced(Scr).Further crossing experiments showed that CasRx induced a down-regulation of Scr transcription in silkworms,which impaired systemic growth of larvae.Overall,this study demonstrated that the CRISPR-Cas13RNA editing system works efficiently in the silkworm,providing a potential alternative approach for RNA manipulation in lepidopteran insects.展开更多
基金supported by grants from National Natural Science Foundation of China(32001571)R&D Program of Beijing Municipal Education Commission(KM202212448003,KM202312448004)+4 种基金Science and Technology Innovation Project of Beijing Vocational College of Agriculture(XY-YF-22-02)Zhongshan Biological Breeding Laboratory(ZSBBL-KY2023-03)China Agriculture Research System of MOF and MARA(CARS-04)Jiangsu Collaborative Innovation Center for Modern Crop Production(JCICMCP)Collaborative Innovation Center for Modern Crop Production co-sponsored by Province and Ministry(CIC-MCP).
文摘Soybean mosaic virus(SMV),an RNA virus,is the most common and destructive pathogenic virus in soybean fields.The newly developed CRISPR/Cas immune system has provided a novel strategy for improving plant resistance to viruses;hence,this study aimed to engineer SMV resistance in soybean using this system.Specifically,multiple sgRNAs were designed to target positive-and/or negative-sense strands of the SMV HC-Pro gene.Subsequently,the corresponding CRISPR/CasRx vectors were constructed and transformed into soybeans.After inoculation with SMV,39.02%,35.77%,and 18.70%of T_(1)plants were confirmed to be highly resistant(HR),resistant(R),and mildly resistant(MR)to SMV,respectively,whereas only 6.50%were identified as susceptible(S).Additionally,qRT-PCR and DAS-ELISA showed that,both at 15 and 30 d post-inoculation(dpi),SMV accumulation significantly decreased or was even undetectable in HR and R plants,followed by MR and S plants.Additionally,the expression level of the CasRx gene varied in almost all T_(1)plants with different resistance level,both at 15 and 30 dpi.Furthermore,when SMV resistance was evaluated in the T_(2)generation,the results were similar to those recorded for the T_(1)generation.These findings provide new insights into the application of the CRISPR/CasRx system for soybean improvement and offer a promising alternative strategy for breeding for resistance to biotic stress that will contribute to the development of SMV-immune soybean germplasm to accelerate progress towards greater soybean crop productivity.
基金supported by the National Key Research and Development Program of China(No.2022YFA1207500)the National Natural Science Foundation of China(No.82394445,82250710174,82161160342,82030067 to D.C.)+1 种基金the Research Grants Council(RGC)of Hong Kong,China(No.HKU-17101821 to W.W.L.and D.C.)the Shenzhen Science and Technology Research Funding(China)(No.JSGGKQTD20210831174330015 to D.C.).
文摘Osteoarthritis(OA)is a chronic degenerative joint disease.Currently,OA is incur-able.Abnormal activation of canonical Wnt/b-catenin or Indian hedgehog(Ihh)signaling could lead to OA development and progression.This study aimed to determine if targeting b-catenin and Ihh signaling could yield an effective therapeutic intervention for OA disease.CRISPR/CasRx is a new RNA interference tool that can precisely and efficiently cleave single-strand RNAs.In this study,we screened CRISPR-derived RNA(crRNA)targeting Ctnnb1 and Smo in vitro and selected two optimal crRNAs for each gene.CasRx-mediated Ctnnb1 and Smo knockdown showed high efficiency and specificity with no obvious off-target effects in vitro.We then per-formed intra-articular injection of selected crRNAs driven by the adeno-associated virus into an OA mouse model.Micro-CT,histological,and histomorphometric analyses were conducted to evaluate the efficacy of CasRx approach on OA treatment.We found that the knockdown of Ctnnb1 and Smo decelerated pathological damage in the keen joint of the experimental OA mouse model.Our findings suggest that CasRx-mediated Ctnnb1 and Smo knockdown could be a potential strategy for OA treatment.
基金the National Key Research and Development Project of China(2019YFA0801500)Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences(CIFMS,2021-I2M-1-008)the National Natural Science Foundation of China(81770308,81900343)。
文摘The RNA editing tool CRISPR-CasRx has provided a platform for a range of transcriptome analysis tools and therapeutic approaches with its broad efficacy and high specificity.To enable the application of CasRx in vivo,we established a Credependent CasRx knock-in mouse.Using these mice,we specifically knocked down the expression of Meis1 and Hoxb13 in cardiomyocytes,which induced cardiac regeneration after myocardial infarction.We also knocked down the lnc RNA Mhrt in cardiomyocytes with the CasRx knock-in mice,causing hypertrophic cardiomyopathy.In summary,we generated a Credependent CasRx knock-in mouse that can efficiently knock down coding gene and lnc RNA expression in specific somatic cells.This in vivo CRISPR-CasRx system is promising for gene function research and disease modeling.
基金supported by the National Natural Science Foundation of China(32070496,32370555)Fundamental Research Funds for the Central Universities(SWU120033)Technology Innovation and Application Development Program of Chongqing(CSTB2024TIADKPX0023)。
文摘The CRISPR-Cas13 system,an RNA-guided editing tool,has emerged as a highly efficient and stable RNA editing technique.Although the CRISPR-Cas13 system has been developed in several insect species,its application in lepidopterans has not yet been reported.In the present study,we evaluated the RNA cleavage activity of the CRISPR-Cas13 system in the silkworm(Bombyx mori),a model lepidopteran insect,both ex vivo and in vivo.We established two stable silkworm BmE cell lines expressing PspCas13b and CasRx,respectively.Further analysis demonstrated that both PspCas13b and CasRx effectively down-regulated the transcription of exogenouslyintroduced target and endogenous genes in these cell lines.In addition,we generated two transgenic silkworm strains,one expressing CasRx and the other expressing RNA-guided CRISPR RNA targeting Sex combs reduced(Scr).Further crossing experiments showed that CasRx induced a down-regulation of Scr transcription in silkworms,which impaired systemic growth of larvae.Overall,this study demonstrated that the CRISPR-Cas13RNA editing system works efficiently in the silkworm,providing a potential alternative approach for RNA manipulation in lepidopteran insects.
