目的利用CRISPR/Cas9基因编辑技术构建斑马鱼map3k15敲除纯合品系,为进一步研究map3k15在肾脏疾病方面的作用提供动物模型。方法1)分析map3k15在斑马鱼中的表达模式;2)利用CRISPR/Cas9基因编辑技术构建斑马鱼map3k15敲除纯合品系;3)观察...目的利用CRISPR/Cas9基因编辑技术构建斑马鱼map3k15敲除纯合品系,为进一步研究map3k15在肾脏疾病方面的作用提供动物模型。方法1)分析map3k15在斑马鱼中的表达模式;2)利用CRISPR/Cas9基因编辑技术构建斑马鱼map3k15敲除纯合品系;3)观察map3k15缺失对斑马鱼表型的影响。结果1)map3k15在斑马鱼前肾中表达,且MAP3K15/Map3k15蛋白在多物种间具有高度保守性;2)成功构建了斑马鱼map3k15-/-突变体,并保留+2 bp及+1 bp 2种突变品系;3)map3k15突变斑马鱼在胚胎发育过程中出现卵黄囊、心包及头部的水肿,且随发育时间延长,症状逐渐加重。结论成功构建了map3k15敲除的纯合斑马鱼品系,为未来研究map3k15在肾脏发育及疾病中的作用提供了重要的模型。展开更多
孕烷X受体(Pregnane X receptor,PXR)是核受体超家族(NRs)中NR1I的重要成员之一,在保护机体免于内源性和外源性物质损伤方面具有重要作用。利用CRISPR/Cas9基因编辑技术建立PXR基因敲除的斑马鱼模型,为研究环境污染物的毒性机理及代谢...孕烷X受体(Pregnane X receptor,PXR)是核受体超家族(NRs)中NR1I的重要成员之一,在保护机体免于内源性和外源性物质损伤方面具有重要作用。利用CRISPR/Cas9基因编辑技术建立PXR基因敲除的斑马鱼模型,为研究环境污染物的毒性机理及代谢过程提供基础模型。根据PXR基因序列,设计gRNA靶位点,体外转录合成gRNA。同时,将设计合成的gRNA与Cas9 mRNA通过显微注射转入斑马鱼受精卵,经孵化、筛选出有效的突变体,并逐代培养杂交筛选出PXR基因敲除突变体。结果表明利用CRISPR/Cas9基因编辑技术成功敲除PXR基因,经测序分析获得稳定的PXR(+4/+4)基因纯合体。展开更多
The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system,belonging to the typeⅡCRISPR/Cas system,is an effective gene-editing tool widely used in different organis...The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system,belonging to the typeⅡCRISPR/Cas system,is an effective gene-editing tool widely used in different organisms,but the size of Streptococcus pyogenes Cas9(SpCas9)is quite large(4.3 kb),which is not convenient for vector delivery.In this study,we used a codon-optimized Staphylococcus aureus Cas9(SaCas9)system to edit the tyrosinase(tyr),oculocutaneous albinismⅡ(oca2),and paired box 6.1(pax6.1)genes in the fish model medaka(Oryzias latipes),in which the size of SaCas9(3.3 kb)is much smaller and the necessary protospacer-adjacent motif(PAM)sequence is 5'-NNGRRT-3'.We also used a transfer RNA(tRNA)-single-guide RNA(sgRNA)system to express the functional sgRNA by transcription either in vivo or in vitro,and the combination of SaCas9 and tRNA-sgRNA was used to edit the tyr gene in the medaka genome.The SaCas9/sgRNA and SaCas9/tRNA-sgRNA systems were shown to edit the medaka genome effectively,while the PAM sequence is an essential part for the efficiency of editing.Besides,tRNA can improve the flexibility of the system by enabling the sgRNA to be controlled by a common promoter such as cytomegalovirus.Moreover,the all-in-one cassette cytomegalovirus(CMV)-SaCas9-tRNA-sgRNA-tRNA is functional in medaka gene editing.Taken together,the codon-optimized SaCas9 system provides an alternative and smaller tool to edit the medaka genome and potentially other fish genomes.展开更多
The whole-genome sequence of the olive flounder(Paralichthys olivaceus)provides a basis for gene functional analyses,which is important for the aquaculture industry.Understanding gene function will help us to select b...The whole-genome sequence of the olive flounder(Paralichthys olivaceus)provides a basis for gene functional analyses,which is important for the aquaculture industry.Understanding gene function will help us to select better economic traits such as fast growth and better culture conditions,which further will increase the aquaculture output.Gene knockout is an important reverse genetics approach for in vivo studies of gene function.In this study,the CRISPR/Cas9 genome editing method with a microinjection system using a simple braked needle was employed in olive flounder.After injection in embryos,green fluorescent protein expression was detected in 40%of larvae.The proportion of normal-hatched larvae was approximately 50%.Different mutations,including short indels and fragment deletions,were found in our test genes gsdf and myomaker.Additionally,we detected more than one mutation in a single larva.In summary,our microinjection technique and CRISPR/Cas9 can be applied to study gene functions in olive flounder.展开更多
Generation of mutants with clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA(mRN...Generation of mutants with clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA(mRNA)or protein and transcribed guide RNA(gRNA).However,the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present.In this study,we employed a poly-transfer RNA(tRNA)-gRNA(PTG)system driven by cytomegalovirus(CMV)promoter to target the medaka(Oryzias latipes)endogenous gene tyrosinase(tyr)or paired box 6.1(pax6.1)and illustrated its function in a medaka cell line and embryos.The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka.This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.展开更多
As a precise and versatile tool for genome manipulation,the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)platform holds promise for modifying fish traits of intere...As a precise and versatile tool for genome manipulation,the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)platform holds promise for modifying fish traits of interest.With the aim of reducing transgene introgression and controlling reproduction,upscaled disease resistance and reproductive intervention in catfish species have been studied to lower the potential environmental risks of the introgression of escapees as transgenic animals.Taking advantage of the CRISPR/Cas9-mediated system,we succeeded in integrating the cathelicidin gene(As-Cath)from an alligator(Alligator sinensis)into the target luteinizing hormone(lh)locus of channel catfish(Ictalurus punctatus)using two delivery systems assisted by double-stranded DNA(dsDNA)and single-stranded oligodeoxynucleotides(ssODNs),respectively.In this study,high knock in(KI)efficiency(22.38%,64/286)but low ontarget events was achieved using the ssODN strategy,whereas adopting a dsDNA as the donor template led to an efficient on-target KI(10.80%,23/213).The on-target KI of As-Cath was instrumental in establishing the lh knockout(LH^(–)_As-Cath^(+))catfish line,which displayed heightened disease resistance and reduced fecundity compared with the wild-type(WT)sibling fish.Furthermore,administration of human chorionic gonadotropin(HCG)and luteinizing hormone-releasing hormone analogue(LHRHa)can restore the reproduction of the transgenic fish line.Overall,we replaced the lh gene with an alligator cathelicidin transgene and then administered hormone therapy to move towards complete reproductive control of diseaseresistant transgenic catfish in an environmentally responsible manner.This strategy not only effectively improves consumer-valued traits but also guards against unwanted introgression,providing a breakthrough in aquaculture genetics to confine fish reproduction and prevent the establishment of transgenic or domestic genotypes in the natural environment.展开更多
文摘目的利用CRISPR/Cas9基因编辑技术构建斑马鱼map3k15敲除纯合品系,为进一步研究map3k15在肾脏疾病方面的作用提供动物模型。方法1)分析map3k15在斑马鱼中的表达模式;2)利用CRISPR/Cas9基因编辑技术构建斑马鱼map3k15敲除纯合品系;3)观察map3k15缺失对斑马鱼表型的影响。结果1)map3k15在斑马鱼前肾中表达,且MAP3K15/Map3k15蛋白在多物种间具有高度保守性;2)成功构建了斑马鱼map3k15-/-突变体,并保留+2 bp及+1 bp 2种突变品系;3)map3k15突变斑马鱼在胚胎发育过程中出现卵黄囊、心包及头部的水肿,且随发育时间延长,症状逐渐加重。结论成功构建了map3k15敲除的纯合斑马鱼品系,为未来研究map3k15在肾脏发育及疾病中的作用提供了重要的模型。
文摘孕烷X受体(Pregnane X receptor,PXR)是核受体超家族(NRs)中NR1I的重要成员之一,在保护机体免于内源性和外源性物质损伤方面具有重要作用。利用CRISPR/Cas9基因编辑技术建立PXR基因敲除的斑马鱼模型,为研究环境污染物的毒性机理及代谢过程提供基础模型。根据PXR基因序列,设计gRNA靶位点,体外转录合成gRNA。同时,将设计合成的gRNA与Cas9 mRNA通过显微注射转入斑马鱼受精卵,经孵化、筛选出有效的突变体,并逐代培养杂交筛选出PXR基因敲除突变体。结果表明利用CRISPR/Cas9基因编辑技术成功敲除PXR基因,经测序分析获得稳定的PXR(+4/+4)基因纯合体。
基金supported by the National Natural Science Foundation of China(Nos.32273127,31771648,and 31672653)the Scientific Research Foundation of Jimei University(No.ZQ2020003),China。
文摘The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system,belonging to the typeⅡCRISPR/Cas system,is an effective gene-editing tool widely used in different organisms,but the size of Streptococcus pyogenes Cas9(SpCas9)is quite large(4.3 kb),which is not convenient for vector delivery.In this study,we used a codon-optimized Staphylococcus aureus Cas9(SaCas9)system to edit the tyrosinase(tyr),oculocutaneous albinismⅡ(oca2),and paired box 6.1(pax6.1)genes in the fish model medaka(Oryzias latipes),in which the size of SaCas9(3.3 kb)is much smaller and the necessary protospacer-adjacent motif(PAM)sequence is 5'-NNGRRT-3'.We also used a transfer RNA(tRNA)-single-guide RNA(sgRNA)system to express the functional sgRNA by transcription either in vivo or in vitro,and the combination of SaCas9 and tRNA-sgRNA was used to edit the tyr gene in the medaka genome.The SaCas9/sgRNA and SaCas9/tRNA-sgRNA systems were shown to edit the medaka genome effectively,while the PAM sequence is an essential part for the efficiency of editing.Besides,tRNA can improve the flexibility of the system by enabling the sgRNA to be controlled by a common promoter such as cytomegalovirus.Moreover,the all-in-one cassette cytomegalovirus(CMV)-SaCas9-tRNA-sgRNA-tRNA is functional in medaka gene editing.Taken together,the codon-optimized SaCas9 system provides an alternative and smaller tool to edit the medaka genome and potentially other fish genomes.
基金the National Natural Sci-ence Foundation of China(Nos.31672636,31772834,and 31972774)the National Key R&D Program of China(Nos.2018YFD0901202 and 2018YFD0900202)the Key Research and Development Program of Shandong Pro-vince,China(No.2019GHY1120070)。
文摘The whole-genome sequence of the olive flounder(Paralichthys olivaceus)provides a basis for gene functional analyses,which is important for the aquaculture industry.Understanding gene function will help us to select better economic traits such as fast growth and better culture conditions,which further will increase the aquaculture output.Gene knockout is an important reverse genetics approach for in vivo studies of gene function.In this study,the CRISPR/Cas9 genome editing method with a microinjection system using a simple braked needle was employed in olive flounder.After injection in embryos,green fluorescent protein expression was detected in 40%of larvae.The proportion of normal-hatched larvae was approximately 50%.Different mutations,including short indels and fragment deletions,were found in our test genes gsdf and myomaker.Additionally,we detected more than one mutation in a single larva.In summary,our microinjection technique and CRISPR/Cas9 can be applied to study gene functions in olive flounder.
基金This study was supported by the National Natural Science Foundation of China(Nos.31771648 and 31672653)the Scientific Research Foundation of Jimei University(No.ZQ2020003)the National Key Basic Research Program of China(No.2013CB967700).
文摘Generation of mutants with clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA(mRNA)or protein and transcribed guide RNA(gRNA).However,the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present.In this study,we employed a poly-transfer RNA(tRNA)-gRNA(PTG)system driven by cytomegalovirus(CMV)promoter to target the medaka(Oryzias latipes)endogenous gene tyrosinase(tyr)or paired box 6.1(pax6.1)and illustrated its function in a medaka cell line and embryos.The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka.This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.
基金supported by USDA Grant(2018-33522-28769)Alabama Agricultural Experiment Station grant(AAES-AIR).
文摘As a precise and versatile tool for genome manipulation,the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)platform holds promise for modifying fish traits of interest.With the aim of reducing transgene introgression and controlling reproduction,upscaled disease resistance and reproductive intervention in catfish species have been studied to lower the potential environmental risks of the introgression of escapees as transgenic animals.Taking advantage of the CRISPR/Cas9-mediated system,we succeeded in integrating the cathelicidin gene(As-Cath)from an alligator(Alligator sinensis)into the target luteinizing hormone(lh)locus of channel catfish(Ictalurus punctatus)using two delivery systems assisted by double-stranded DNA(dsDNA)and single-stranded oligodeoxynucleotides(ssODNs),respectively.In this study,high knock in(KI)efficiency(22.38%,64/286)but low ontarget events was achieved using the ssODN strategy,whereas adopting a dsDNA as the donor template led to an efficient on-target KI(10.80%,23/213).The on-target KI of As-Cath was instrumental in establishing the lh knockout(LH^(–)_As-Cath^(+))catfish line,which displayed heightened disease resistance and reduced fecundity compared with the wild-type(WT)sibling fish.Furthermore,administration of human chorionic gonadotropin(HCG)and luteinizing hormone-releasing hormone analogue(LHRHa)can restore the reproduction of the transgenic fish line.Overall,we replaced the lh gene with an alligator cathelicidin transgene and then administered hormone therapy to move towards complete reproductive control of diseaseresistant transgenic catfish in an environmentally responsible manner.This strategy not only effectively improves consumer-valued traits but also guards against unwanted introgression,providing a breakthrough in aquaculture genetics to confine fish reproduction and prevent the establishment of transgenic or domestic genotypes in the natural environment.
