目的:探索CRISPR干扰(CRISPR interference,C R I S P R i)能否实现在体抑制肝脏m i R-122表达.方法:针对m i R-1 2 2启动子区设计sg RNA(sg T1和sg T2),并分别将其与无DNA切割活性仅保留识别活性的d Cas9-KRAB载体通过尾静脉流体力学...目的:探索CRISPR干扰(CRISPR interference,C R I S P R i)能否实现在体抑制肝脏m i R-122表达.方法:针对m i R-1 2 2启动子区设计sg RNA(sg T1和sg T2),并分别将其与无DNA切割活性仅保留识别活性的d Cas9-KRAB载体通过尾静脉流体力学法注射到8-10 wk龄小鼠,注射1、2、4 wk后通过实时荧光定量PCR(quantitative real-time PCR,q RT-PCR)方法检测肝脏mmu-mi R-122的表达;设计不同的sg RNA浓度梯度,探索CRISPRi在体抑制肝脏mi R-122表达是否存在剂量依赖性;通过q RT-PCR及Western blot方法检测肝脏mi R-122靶分子HOMX1和Cyclin G1的表达变化.结果:在注射1 wk和2 wk后,sg T1介导的C R I S P R i在体抑制肝脏m i R-122的表达水平分别为23%(P<0.05)和16%(P<0.05);随sg RNA的剂量升高,肝脏mi R-122表达降低,当lenti Guide-Puro-sg T1质粒为120?g时,可将mi R-122的表达抑制约30%;CRIPSRi在体抑制肝脏mi R-122表达的同时,上调了mi R-122下游靶分子HMOX1和Cyclin G1的表达.结论:本研究利用CRISPRi实现了在体抑制肝脏mi R-122的表达,为抗丙型肝炎病毒(hepatitis C virus)的在体治疗提供了新的策略.展开更多
Non-homologous end-joining(NHEJ) is a predominant pathway for the repair of DNA double-strand breaks(DSB). It inhibits the efficiency of homologous recombination(HR) by competing for DSB targets. To improve the effici...Non-homologous end-joining(NHEJ) is a predominant pathway for the repair of DNA double-strand breaks(DSB). It inhibits the efficiency of homologous recombination(HR) by competing for DSB targets. To improve the efficiency of HR, multiple CRISPR interference(CRISPRi) and Natronobacterium gregoryi Argonaute(NgAgo) interference(NgAgoi) systems have been designed for the knockdown of NHEJ key molecules, KU70, KU80, polynucleotide kinase/phosphatase(PNKP), DNA ligase IV(LIG4), and NHEJ1. Suppression of KU70 and KU80 by CRISPRi dramatically promoted(P<0.05) the efficiency of HR to 1.85-and 1.58-fold, respectively, whereas knockdown of PNKP, LIG4, and NHEJ1 repair factors did not significantly increase(P>0.05) HR efficiency. Interestingly, although the NgAgoi system significantly suppressed(P<0.05) KU70, KU80, PNKP, LIG4, and NHEJ1 expression, it did not improve(P>0.05) HR efficiency in primary fetal fibroblasts. Our result showed that both NgAgo and catalytically inactive Cas9(dCas9) could interfere with the expression of target genes, but the downstream factors appear to be more active following CRISPR-mediated interference than that of NgAgo.展开更多
<div style="text-align:justify;"> Neurotrophic factors, as well as their receptors are key players in the formation and development of the central nervous system. Like the sculptor’s incisor, they for...<div style="text-align:justify;"> Neurotrophic factors, as well as their receptors are key players in the formation and development of the central nervous system. Like the sculptor’s incisor, they form the neural networks and circuits of the future organism. The neurotrophic growth factor receptor p75ngfr interacts with sortilin, serves as a receptor for proform of neurotrophic factors and exhibits a proapoptotic effect in developing neurons—dorsal root ganglia neurons and brainstem norepinephrine neurons. p75ngfr is highly expressed in Locus Coeruleus norepinephrine neurons. Therefore, an important task for developing further methods of CNS gene therapy is the development of tools and molecular methods for suppressing p75ngfr expression in norepinephrine neurons. For this purpose, we’ve developed improved dCas9 vectors with Suntag system to suppress gene expression and enhance methylation of CpG islands. We used 10 times repetitive GCN peptide that were fused to dCas9. Single chain antibody against GCN peptide was fused to KRAB repressor or Dnmt3a catalytic domain. Expression specificity was achieved by using a promoter consisting of 8 repeated phox2a/2b binding sites. In this work, we’ve tested a set of guide RNAs targeting p75ngfr cpg island in the promoter. Usage of Suntag system led us to the conclusion that topological orientation and length of the final complex could influence on p75ngfr antisense transcript expression, and that sequence was established in the rat P3 brainstem. </div>展开更多
Objective:Pancreatic ductal adenocarcinoma(PDAC)is a highly malignant gastrointestinal cancer with a 5-year survival rate of only 9%.Of PDAC patients,15%-20%are eligible for radical surgery.Gemcitabine is an important...Objective:Pancreatic ductal adenocarcinoma(PDAC)is a highly malignant gastrointestinal cancer with a 5-year survival rate of only 9%.Of PDAC patients,15%-20%are eligible for radical surgery.Gemcitabine is an important chemotherapeutic agent for patients with PDAC;however,the efficacy of gemcitabine is limited due to resistance.Therefore,reducing gemcitabine resistance is essential for improving survival of patients with PDAC.Identifying the key target that determines gemcitabine resistance in PDAC and reversing gemcitabine resistance using target inhibitors in combination with gemcitabine are crucial steps in the quest to improve survival prognosis in patients with PDAC.Methods:We constructed a human genome-wide CRISPRa/dCas 9 overexpression library in PDAC cell lines to screen key targets of drug resistance based on sgRNA abundance and enrichment.Then,co-IP,ChIP,ChIP-seq,transcriptome sequencing,and qPCR were used to determine the specific mechanism by which phospholipase D1(PLD1)confers resistance to gemcitabine.Results:PLD1 combines with nucleophosmin 1(NPM1)and triggers NPM1 nuclear translocation,where NPM1 acts as a transcription factor to upregulate interleukin 7 receptor(IL7R)expression.Upon interleukin 7(IL-7)binding,IL7R activates the JAK1/STAT5 signaling pathway to increase the expression of the anti-apoptotic protein,BCL-2,and induce gemcitabine resistance.The PLD1 inhibitor,Vu0155069,targets PLD1 to induce apoptosis in gemcitabine-resistant PDAC cells.Conclusions:PLD1 is an enzyme that has a critical role in PDAC-associated gemcitabine resistance through a non-enzymatic interaction with NPM1,further promoting the downstream JAK1/STAT5/Bcl-2 pathway.Inhibiting any of the participants of this pathway can increase gemcitabine sensitivity.展开更多
Background:The ability to generate functional hepatocytes without relying on donor liver organs holds significant therapeutic promise in the fields of regenerative medicine and potential liver disease treatments.Clust...Background:The ability to generate functional hepatocytes without relying on donor liver organs holds significant therapeutic promise in the fields of regenerative medicine and potential liver disease treatments.Clustered regularly interspaced short palindromic repeats(CRISPR)activator(CRISPRa)is a powerful tool that can conveniently and efficiently activate the expression of multiple endogenous genes simultaneously,providing a new strategy for cell fate determination.The main purpose of this study is to explore the feasibility of applying CRISPRa for hepatocyte reprogramming and its application in the treatment of mouse liver fibrosis.Method:The differentiation of mouse embryonic fibroblasts(MEFs)into functional induced hepatocyte-like cells(iHeps)was achieved by utilizing the CRISPRa synergistic activation mediator(SAM)system,which drove the combined expression of three endogenous transcription factors-Gata4,Foxa3,and Hnf1a-or alternatively,the expression of two transcription factors,Gata4 and Foxa3.In vivo,we injected adeno-associated virus serotype 6(AAV6)carrying the CRISPRa SAM system into liver fibrotic Col1a1-Cre^(ER);Cas9^(fl/fl)mice,effectively activating the expression of endogenous Gata4 and Foxa3 in fibroblasts.The endogenous transcriptional activation of genes was confirmed using real-time quantitative polymerase chain reaction(RT-qPCR)and RNA-seq,and the morphology and characteristics of the induced hepatocytes were observed through microscopy.The level of hepatocyte reprogramming in vivo is detected by immunofluorescence staining,while the improvement of liver fibrosis is evaluated through Sirius red staining,alpha-smooth muscle actin(α-SMA)immunofluorescence staining,and blood alanine aminotransferase(ALT)examination.Results:Activation of only two factors,Gata4 and Foxa3,via CRISPRa was sufficient to successfully induce the transformation of MEFs into iHeps.These iHeps could be expanded in vitro and displayed functional characteristics similar to those of mature hepatocytes,such as drug metabolism and glycogen storage.Additionally,AAV6-based delivery of the CRISPRa SAM system effectively induced the hepatic reprogramming from fibroblasts in mice with live fibrosis.After 8 weeks of induction,the reprogrammed hepatocytes comprised 0.87%of the total hepatocyte population in the mice,significantly reducing liver fibrosis.Conclusion:CRISPRa-induced hepatocyte reprogramming may be a promising strategy for generating functional hepatocytes and treating liver fibrosis caused by hepatic diseases.展开更多
文摘目的:探索CRISPR干扰(CRISPR interference,C R I S P R i)能否实现在体抑制肝脏m i R-122表达.方法:针对m i R-1 2 2启动子区设计sg RNA(sg T1和sg T2),并分别将其与无DNA切割活性仅保留识别活性的d Cas9-KRAB载体通过尾静脉流体力学法注射到8-10 wk龄小鼠,注射1、2、4 wk后通过实时荧光定量PCR(quantitative real-time PCR,q RT-PCR)方法检测肝脏mmu-mi R-122的表达;设计不同的sg RNA浓度梯度,探索CRISPRi在体抑制肝脏mi R-122表达是否存在剂量依赖性;通过q RT-PCR及Western blot方法检测肝脏mi R-122靶分子HOMX1和Cyclin G1的表达变化.结果:在注射1 wk和2 wk后,sg T1介导的C R I S P R i在体抑制肝脏m i R-122的表达水平分别为23%(P<0.05)和16%(P<0.05);随sg RNA的剂量升高,肝脏mi R-122表达降低,当lenti Guide-Puro-sg T1质粒为120?g时,可将mi R-122的表达抑制约30%;CRIPSRi在体抑制肝脏mi R-122表达的同时,上调了mi R-122下游靶分子HMOX1和Cyclin G1的表达.结论:本研究利用CRISPRi实现了在体抑制肝脏mi R-122的表达,为抗丙型肝炎病毒(hepatitis C virus)的在体治疗提供了新的策略.
基金supported by the National Science and Technology Major Project for Breeding of New Transgenic Organisms, China (2016ZX08006002)the Guangdong Province "Flying Sail Program" Postdoctoral Foundation, China (2016)
文摘Non-homologous end-joining(NHEJ) is a predominant pathway for the repair of DNA double-strand breaks(DSB). It inhibits the efficiency of homologous recombination(HR) by competing for DSB targets. To improve the efficiency of HR, multiple CRISPR interference(CRISPRi) and Natronobacterium gregoryi Argonaute(NgAgo) interference(NgAgoi) systems have been designed for the knockdown of NHEJ key molecules, KU70, KU80, polynucleotide kinase/phosphatase(PNKP), DNA ligase IV(LIG4), and NHEJ1. Suppression of KU70 and KU80 by CRISPRi dramatically promoted(P<0.05) the efficiency of HR to 1.85-and 1.58-fold, respectively, whereas knockdown of PNKP, LIG4, and NHEJ1 repair factors did not significantly increase(P>0.05) HR efficiency. Interestingly, although the NgAgoi system significantly suppressed(P<0.05) KU70, KU80, PNKP, LIG4, and NHEJ1 expression, it did not improve(P>0.05) HR efficiency in primary fetal fibroblasts. Our result showed that both NgAgo and catalytically inactive Cas9(dCas9) could interfere with the expression of target genes, but the downstream factors appear to be more active following CRISPR-mediated interference than that of NgAgo.
文摘<div style="text-align:justify;"> Neurotrophic factors, as well as their receptors are key players in the formation and development of the central nervous system. Like the sculptor’s incisor, they form the neural networks and circuits of the future organism. The neurotrophic growth factor receptor p75ngfr interacts with sortilin, serves as a receptor for proform of neurotrophic factors and exhibits a proapoptotic effect in developing neurons—dorsal root ganglia neurons and brainstem norepinephrine neurons. p75ngfr is highly expressed in Locus Coeruleus norepinephrine neurons. Therefore, an important task for developing further methods of CNS gene therapy is the development of tools and molecular methods for suppressing p75ngfr expression in norepinephrine neurons. For this purpose, we’ve developed improved dCas9 vectors with Suntag system to suppress gene expression and enhance methylation of CpG islands. We used 10 times repetitive GCN peptide that were fused to dCas9. Single chain antibody against GCN peptide was fused to KRAB repressor or Dnmt3a catalytic domain. Expression specificity was achieved by using a promoter consisting of 8 repeated phox2a/2b binding sites. In this work, we’ve tested a set of guide RNAs targeting p75ngfr cpg island in the promoter. Usage of Suntag system led us to the conclusion that topological orientation and length of the final complex could influence on p75ngfr antisense transcript expression, and that sequence was established in the rat P3 brainstem. </div>
基金supported by the National Key Research and Development Program of China(Grant No.2021YFA1201100)the National Natural Science Foundation of China(Grant Nos.82103006,82030092,81720108028,82072657,82072716,82103003,82173295,81871968,81871978,82072691,and 82103222)+1 种基金the Tianjin Hygiene Healthy Science and Technology Project(Grant No.TJWJ2022MS007)the Science&Technology Development Fund of Tianjin Education Commission for Higher Education(Grant No.2020KJ141).
文摘Objective:Pancreatic ductal adenocarcinoma(PDAC)is a highly malignant gastrointestinal cancer with a 5-year survival rate of only 9%.Of PDAC patients,15%-20%are eligible for radical surgery.Gemcitabine is an important chemotherapeutic agent for patients with PDAC;however,the efficacy of gemcitabine is limited due to resistance.Therefore,reducing gemcitabine resistance is essential for improving survival of patients with PDAC.Identifying the key target that determines gemcitabine resistance in PDAC and reversing gemcitabine resistance using target inhibitors in combination with gemcitabine are crucial steps in the quest to improve survival prognosis in patients with PDAC.Methods:We constructed a human genome-wide CRISPRa/dCas 9 overexpression library in PDAC cell lines to screen key targets of drug resistance based on sgRNA abundance and enrichment.Then,co-IP,ChIP,ChIP-seq,transcriptome sequencing,and qPCR were used to determine the specific mechanism by which phospholipase D1(PLD1)confers resistance to gemcitabine.Results:PLD1 combines with nucleophosmin 1(NPM1)and triggers NPM1 nuclear translocation,where NPM1 acts as a transcription factor to upregulate interleukin 7 receptor(IL7R)expression.Upon interleukin 7(IL-7)binding,IL7R activates the JAK1/STAT5 signaling pathway to increase the expression of the anti-apoptotic protein,BCL-2,and induce gemcitabine resistance.The PLD1 inhibitor,Vu0155069,targets PLD1 to induce apoptosis in gemcitabine-resistant PDAC cells.Conclusions:PLD1 is an enzyme that has a critical role in PDAC-associated gemcitabine resistance through a non-enzymatic interaction with NPM1,further promoting the downstream JAK1/STAT5/Bcl-2 pathway.Inhibiting any of the participants of this pathway can increase gemcitabine sensitivity.
基金National Key Research and Development Program of China(No.2019YFA0801500)National High Level Hospital Clinical Research Funding(No.2023-GSP-ZD-2-01)Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences(No.2021-I2M-1-008)
文摘Background:The ability to generate functional hepatocytes without relying on donor liver organs holds significant therapeutic promise in the fields of regenerative medicine and potential liver disease treatments.Clustered regularly interspaced short palindromic repeats(CRISPR)activator(CRISPRa)is a powerful tool that can conveniently and efficiently activate the expression of multiple endogenous genes simultaneously,providing a new strategy for cell fate determination.The main purpose of this study is to explore the feasibility of applying CRISPRa for hepatocyte reprogramming and its application in the treatment of mouse liver fibrosis.Method:The differentiation of mouse embryonic fibroblasts(MEFs)into functional induced hepatocyte-like cells(iHeps)was achieved by utilizing the CRISPRa synergistic activation mediator(SAM)system,which drove the combined expression of three endogenous transcription factors-Gata4,Foxa3,and Hnf1a-or alternatively,the expression of two transcription factors,Gata4 and Foxa3.In vivo,we injected adeno-associated virus serotype 6(AAV6)carrying the CRISPRa SAM system into liver fibrotic Col1a1-Cre^(ER);Cas9^(fl/fl)mice,effectively activating the expression of endogenous Gata4 and Foxa3 in fibroblasts.The endogenous transcriptional activation of genes was confirmed using real-time quantitative polymerase chain reaction(RT-qPCR)and RNA-seq,and the morphology and characteristics of the induced hepatocytes were observed through microscopy.The level of hepatocyte reprogramming in vivo is detected by immunofluorescence staining,while the improvement of liver fibrosis is evaluated through Sirius red staining,alpha-smooth muscle actin(α-SMA)immunofluorescence staining,and blood alanine aminotransferase(ALT)examination.Results:Activation of only two factors,Gata4 and Foxa3,via CRISPRa was sufficient to successfully induce the transformation of MEFs into iHeps.These iHeps could be expanded in vitro and displayed functional characteristics similar to those of mature hepatocytes,such as drug metabolism and glycogen storage.Additionally,AAV6-based delivery of the CRISPRa SAM system effectively induced the hepatic reprogramming from fibroblasts in mice with live fibrosis.After 8 weeks of induction,the reprogrammed hepatocytes comprised 0.87%of the total hepatocyte population in the mice,significantly reducing liver fibrosis.Conclusion:CRISPRa-induced hepatocyte reprogramming may be a promising strategy for generating functional hepatocytes and treating liver fibrosis caused by hepatic diseases.
