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
Gene expression regulation, including loss-of-function and gain-of-function assays, is a powerful method to study developmental and disease mechanisms. Drosophila melanogaster is an ideal model system particularly wel...Gene expression regulation, including loss-of-function and gain-of-function assays, is a powerful method to study developmental and disease mechanisms. Drosophila melanogaster is an ideal model system particularly well-equipped with many genetic tools. In this review, we describe and discuss the gene expression regulation techniques recently developed and their applications, including the CRISPR/Cas9-triggered heritable mutation system, CRISPR/dCas9-based transcriptional activation(CRISPRa) system,and CRISPR/dCas9-based transcriptional repression(CRISPRi) system, as well as the next-generation transgenic RNAi system. The main purpose of this review is to provide the fly research community with an updated summary of newly developed gene expression regulation techniques and help the community to select appropriate methods and optimize the research strategy.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
LAMA2-related congenital muscular dystrophy(LAMA2-CMD),characterized by laminin-α2 deficiency,is debilitating and ultimately fatal.To date,no effective therapy has been clinically available.Laminin-a1,which shares si...LAMA2-related congenital muscular dystrophy(LAMA2-CMD),characterized by laminin-α2 deficiency,is debilitating and ultimately fatal.To date,no effective therapy has been clinically available.Laminin-a1,which shares significant similarities with laminin-a2,has been proven as a viable compensatory modifier.To evaluate its clinical applicability,we establish a Lama2 exon-3-deletion mouse model(dy^(H)/dy^(H)).The dy^(H)/dy^(H)mice exhibit early lethality and typical LAMA2-CMD phenotypes,allowing the evaluation of various endpoints.In dy^(H)/dy^(H)mice treated with synergistic activation mediator-based CRISPRa-mediated Lama1 upregulation,a nearly doubled median survival is observed,as well as improvements in weight and grip.Significant therapeutical effects are revealed by MRl,serum biochemical indices,and muscle pathology studies.Treating LAMA2-CMD with LAMA1 upregulation is feasible,and early intervention can alleviate symptoms and extend lifespan.Additionally,we reveal the limitations of LAMA1 upregulation,including high-dose mortality and non-sustained expression,which require further optimization in future studies.展开更多
Microalgae,a diverse group of photosynthetic microorganisms with substantial ecological and economic sig-nificance,possess great potential for bioenergy production and high-value bioproduct synthesis.However,largescal...Microalgae,a diverse group of photosynthetic microorganisms with substantial ecological and economic sig-nificance,possess great potential for bioenergy production and high-value bioproduct synthesis.However,largescale industrial application remains constrained by the low efficiency and irreversibility of conventional geneediting approaches.This review systematically examines the epigenetic regulatory mechanisms in microalgae,emphasizing recent advances in epigenome-editing technologies such as CRISPR/dCas(Clustered Regularly Interspaced Short Palindromic Repeats/dead CRISPR-associated protein 9)systems including CRISPR activation(CRISPRa)and CRISPR interference(CRISPRi).These approaches enable reversible regulation of gene expression without altering the DNA sequence by precisely modifying epigenetic marks such as DNA methylation and histone modifications.Despite ongoing challenges related to delivery efficiency and off-target effects,epigenome editing represents a promising precision-regulation strategy that may overcome longstanding barriers in microalgal genetic improvement and accelerate their industrialization.展开更多
Immune checkpoint blockade(ICB)therapy,which has revolutionized cancer treatment,has been approved for the treatment of triple-negative breast cancer(TNBC).Unfortunately,most patients with TNBC are either not eligible...Immune checkpoint blockade(ICB)therapy,which has revolutionized cancer treatment,has been approved for the treatment of triple-negative breast cancer(TNBC).Unfortunately,most patients with TNBC are either not eligible for treatment or exhibit resistance,resulting in limited overall survival benefits.There is an urgent need to elucidate the mechanisms of resistance and enhance therapeutic efficacy.Here,via CRISPR activation(CRISPRa)screening,we identified family with sequence similarity 114 member A1(FAM114A1)as a key mediator of immune evasion and ICB resistance in TNBC.Mechanistically,FAM114A1 binds p85αto disrupt the p85α/p110αprotein complex,thus activating the PI3K/AKT pathway and simultaneously preventing condensate formation of E2F Transcription Factor 4(E2F4)to promote E2F4-driven Metadherin(MTDH)transcription.Upregulation of these FAM114A1-mediated pathways suppresses tumor antigen presentation and consequently attenuates antitumor immunity in TNBC.Moreover,targeting FAM114A1 improves the therapeutic effectiveness of anti-PD-1 therapy in mouse models,and a FAM114A1-based signature shows strong predictive performance for identifying patients with TNBC who may benefit from ICB.Collectively,our findings not only reveal that FAM114A1 is an immune evasion driver but also highlight it as a promising biomarker and therapeutic target.Our study provides new insights into TNBC immune evasion and outlines a potential avenue to improve the effectiveness of ICB.展开更多
Clustered regularly interspaced short palindromic repeats(CRISPR)-Cas systems can be engineered as programmable transcription factors to either activate(CRISPRa)or inhibit transcription.Apomixis is extremely valuable ...Clustered regularly interspaced short palindromic repeats(CRISPR)-Cas systems can be engineered as programmable transcription factors to either activate(CRISPRa)or inhibit transcription.Apomixis is extremely valuable for the seed industry in breeding clonal seeds with pure genetic backgrounds.We report here a CRISPR/dCas9-based toolkit equippedwith dCas9-VP64 andMS2-p65-HSF1 effectors that may specifically target genes with high activation capability.We explored the application of in vivo CRISPRa targeting of maize BABY BOOM2(ZmBBM2),acting as a fertilization checkpoint,as a means to engineer parthenogenesis.We detected ZmBBM2 transcripts only in egg cells but not in other maternal gametic cells.Activation of ZmBBM2 in egg cells in vivo caused maternal cell-autonomous parthenogenesis to produce haploid seeds.Our work provides a highly specific gene-activation CRISPRa technology for target cells and verifies its application for parthenogenesis induction in maize.展开更多
The outbreak of coronavirus disease 2019(COVID-19) caused by SARS-CoV-2 has created a global health crisis. SARS-CoV-2 infects varieties of tissues where the known receptor ACE2 is low or almost absent, suggesting the...The outbreak of coronavirus disease 2019(COVID-19) caused by SARS-CoV-2 has created a global health crisis. SARS-CoV-2 infects varieties of tissues where the known receptor ACE2 is low or almost absent, suggesting the existence of alternative viral entry pathways. Here, we performed a genome-wide barcoded-CRISPRa screen to identify novel host factors that enable SARS-CoV-2 infection. Beyond known host proteins, i.e., ACE2, TMPRSS2, and NRP1, we identified multiple host components,among which LDLRAD3, TMEM30A, and CLEC4G were confirmed as functional receptors for SARS-CoV-2. All these membrane proteins bind directly to spike’s N-terminal domain(NTD). Their essential and physiological roles have been confirmed in either neuron or liver cells. In particular, LDLRAD3 and CLEC4G mediate SARS-CoV-2 entry and infection in an ACE2-independent fashion. The identification of the novel receptors and entry mechanisms could advance our understanding of the multiorgan tropism of SARS-CoV-2, and may shed light on the development of COVID-19 countermeasures.展开更多
α-Pinene is an important monoterpene,which is widely used as a flavoring agent and in fragrances,pharmaceuticals and biofuels.Although an evolved strain Escherichia coli YZFP,which had higher tolerance to pinene and ...α-Pinene is an important monoterpene,which is widely used as a flavoring agent and in fragrances,pharmaceuticals and biofuels.Although an evolved strain Escherichia coli YZFP,which had higher tolerance to pinene and titer,has been successfully used to produce high levels of pinene,the pinene titer is much lower than that of hemiterpene(isoprene)and sesquiterpenes(farnesene)to date.Moreover,the overall cellular physiological and metabolic changes caused by higher tolerance to pinene and overproduction of pinene remains unclear.To reveal the mechanism of Escherichia coli YZFP with the higher tolerance to pinene and titer,a comparative genomics and transcriptional level analyses combining with CRISPR activation(CRISPRa)and interference(CRISPRi)were carried out.The results show that the tolerance to pinene and the overproduction of pinene in E.coli may be associated with:1)the mutations of the DXP pathway genes,the rpoA and some membrane protein genes,and their upregulations of transcription levels;and 2)the mutations of some genes and their downregulation of transcriptional levels.These comparative omics analyses provided some genetic modification strategies to further improve pinene production.Overexpression of the mutated cbpA,tabA,pitA,rpoA,sufBCDS,mutS,ispH,oppF,dusB,dnaK,dxs,dxr and flgFGH genes further improved pinene production.This study also demonstrated that combining comparative omics analysis with CRISPRa and CRISPRi is an efficient technology to quickly find a new metabolic engineering strategy.展开更多
CRISPR-Cas12a genome engineering systems have been widely used in plant research and crop breeding.To date,the performance and use of anti-CRISPR-Cas12a systems have not been fully established in plants.Here,we conduc...CRISPR-Cas12a genome engineering systems have been widely used in plant research and crop breeding.To date,the performance and use of anti-CRISPR-Cas12a systems have not been fully established in plants.Here,we conduct in silico analysis to identify putative anti-CRISPR systems for Cas12a.These putative anti-CRISPR proteins,along with known anti-CRISPR proteins,are assessed for their ability to inhibit Cas12a cleavage activity in vivo and in planta.Among all anti-CRISPR proteins tested,AcrVA1 shows robust inhibition of Mb2Cas12a and LbCas12a in E.coli.Further tests show that Acr VA1 inhibits LbCas12a mediated genome editing in rice protoplasts and stable transgenic lines.Impressively,co-expression of Acr VA1 mitigates off-target effects by CRISPR-LbCas12a,as revealed by whole genome sequencing.In addition,transgenic plants expressing AcrVA1 exhibit different levels of inhibition to LbCas12a mediated genome editing,representing a novel way of fine-tuning genome editing efficiency.By controlling temporal and spatial expression of AcrVA1,we show that inducible and tissue specific genome editing can be achieved in plants.Furthermore,we demonstrate that AcrVA1 also inhibits Lb Cas12a-based CRISPR activation(CRISPRa)and based on this principle we build logic gates to turn on and off target genes in plant cells.Together,we have established an efficient anti-CRISPR-Cas12a system in plants and demonstrate its versatile applications in mitigating off-target effects,finetuning genome editing efficiency,achieving spatial-temporal control of genome editing,and generating synthetic logic gates for controlling target gene expression in plant cells.展开更多
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.
基金supported by the National Natural Science Foundation of China(Nos.31571320,31872818,and 31801079)the National Key Technology Research and Development Program of the Ministry of Science and Technology of China(2016YFE0113700)the China Postdoctoral Science Foundation(2017M620747)
文摘Gene expression regulation, including loss-of-function and gain-of-function assays, is a powerful method to study developmental and disease mechanisms. Drosophila melanogaster is an ideal model system particularly well-equipped with many genetic tools. In this review, we describe and discuss the gene expression regulation techniques recently developed and their applications, including the CRISPR/Cas9-triggered heritable mutation system, CRISPR/dCas9-based transcriptional activation(CRISPRa) system,and CRISPR/dCas9-based transcriptional repression(CRISPRi) system, as well as the next-generation transgenic RNAi system. The main purpose of this review is to provide the fly research community with an updated summary of newly developed gene expression regulation techniques and help the community to select appropriate methods and optimize the research strategy.
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This study received support from the following grants:National Natural Science Foundation of China(82171393 to H.X.)National High Level Hospital Clinical Research Funding(High Quality Clinical Research Project of Peking University First Hospital)(2022CR69 to H.X.)+4 种基金Natural Science Foundation of Beijing Municipality(7212116 to H.X.)National Key Research and Development Program of China(2016YFC0901505 to H.X.)Beijing Key Laboratory of Molecular Diagnosis and Study on Pediatric Genetic Diseases(BZ0317 to H.X.)Research Foundation for Youth Talents of the First Affiliated Hospital of Nanchang University(YFYPY202223 to D.T.)Natural Science Foundation of Beijing Municipality(7242149 to H.L.).
文摘LAMA2-related congenital muscular dystrophy(LAMA2-CMD),characterized by laminin-α2 deficiency,is debilitating and ultimately fatal.To date,no effective therapy has been clinically available.Laminin-a1,which shares significant similarities with laminin-a2,has been proven as a viable compensatory modifier.To evaluate its clinical applicability,we establish a Lama2 exon-3-deletion mouse model(dy^(H)/dy^(H)).The dy^(H)/dy^(H)mice exhibit early lethality and typical LAMA2-CMD phenotypes,allowing the evaluation of various endpoints.In dy^(H)/dy^(H)mice treated with synergistic activation mediator-based CRISPRa-mediated Lama1 upregulation,a nearly doubled median survival is observed,as well as improvements in weight and grip.Significant therapeutical effects are revealed by MRl,serum biochemical indices,and muscle pathology studies.Treating LAMA2-CMD with LAMA1 upregulation is feasible,and early intervention can alleviate symptoms and extend lifespan.Additionally,we reveal the limitations of LAMA1 upregulation,including high-dose mortality and non-sustained expression,which require further optimization in future studies.
基金supported by Natural Science Foundation of China(32160020 and 32360022)the fund of Innovation Platform for Academicians of Hainan Province,China(YSPTZX202130).
文摘Microalgae,a diverse group of photosynthetic microorganisms with substantial ecological and economic sig-nificance,possess great potential for bioenergy production and high-value bioproduct synthesis.However,largescale industrial application remains constrained by the low efficiency and irreversibility of conventional geneediting approaches.This review systematically examines the epigenetic regulatory mechanisms in microalgae,emphasizing recent advances in epigenome-editing technologies such as CRISPR/dCas(Clustered Regularly Interspaced Short Palindromic Repeats/dead CRISPR-associated protein 9)systems including CRISPR activation(CRISPRa)and CRISPR interference(CRISPRi).These approaches enable reversible regulation of gene expression without altering the DNA sequence by precisely modifying epigenetic marks such as DNA methylation and histone modifications.Despite ongoing challenges related to delivery efficiency and off-target effects,epigenome editing represents a promising precision-regulation strategy that may overcome longstanding barriers in microalgal genetic improvement and accelerate their industrialization.
基金supported by grants from the National Natural Science Foundation of China(2021hwyq55 and 82472950 to M.Shen,32270745 to Y.Lu)the Natural Science Foundation of Shanghai(23ZR1466500 to Y.Lu)+1 种基金the Shanghai Municipal Health Commission(2022YQ067 to Y.Lu)supported by the Human Phenome Data Center of Fudan University。
文摘Immune checkpoint blockade(ICB)therapy,which has revolutionized cancer treatment,has been approved for the treatment of triple-negative breast cancer(TNBC).Unfortunately,most patients with TNBC are either not eligible for treatment or exhibit resistance,resulting in limited overall survival benefits.There is an urgent need to elucidate the mechanisms of resistance and enhance therapeutic efficacy.Here,via CRISPR activation(CRISPRa)screening,we identified family with sequence similarity 114 member A1(FAM114A1)as a key mediator of immune evasion and ICB resistance in TNBC.Mechanistically,FAM114A1 binds p85αto disrupt the p85α/p110αprotein complex,thus activating the PI3K/AKT pathway and simultaneously preventing condensate formation of E2F Transcription Factor 4(E2F4)to promote E2F4-driven Metadherin(MTDH)transcription.Upregulation of these FAM114A1-mediated pathways suppresses tumor antigen presentation and consequently attenuates antitumor immunity in TNBC.Moreover,targeting FAM114A1 improves the therapeutic effectiveness of anti-PD-1 therapy in mouse models,and a FAM114A1-based signature shows strong predictive performance for identifying patients with TNBC who may benefit from ICB.Collectively,our findings not only reveal that FAM114A1 is an immune evasion driver but also highlight it as a promising biomarker and therapeutic target.Our study provides new insights into TNBC immune evasion and outlines a potential avenue to improve the effectiveness of ICB.
基金supported by the National Science Foundation of China(32001551 and 31771808)the China Postdoctoral Science Foundation(2020M680779)+1 种基金the Agricultural Science and Technology Innovation Program of the CAAS(S2022ZD03)Hainan Yazhou Bay Seed Laboratory(B21HJ0215).
文摘Clustered regularly interspaced short palindromic repeats(CRISPR)-Cas systems can be engineered as programmable transcription factors to either activate(CRISPRa)or inhibit transcription.Apomixis is extremely valuable for the seed industry in breeding clonal seeds with pure genetic backgrounds.We report here a CRISPR/dCas9-based toolkit equippedwith dCas9-VP64 andMS2-p65-HSF1 effectors that may specifically target genes with high activation capability.We explored the application of in vivo CRISPRa targeting of maize BABY BOOM2(ZmBBM2),acting as a fertilization checkpoint,as a means to engineer parthenogenesis.We detected ZmBBM2 transcripts only in egg cells but not in other maternal gametic cells.Activation of ZmBBM2 in egg cells in vivo caused maternal cell-autonomous parthenogenesis to produce haploid seeds.Our work provides a highly specific gene-activation CRISPRa technology for target cells and verifies its application for parthenogenesis induction in maize.
基金supported by funds from the National Key R&D Program of China (2020YFA0707800 to W.W., 2020YFA0707600 to Z.Z.)Beijing Municipal Science & Technology Commission (Z181100001318009)+4 种基金the National Natural Science Foundation of China (31930016)Beijing Advanced Innovation Center for Genomics at Peking University and the Peking-Tsinghua Center for Life Sciences (to W.W.)the National Natural Science Foundation of China (31870893)the National Major Science & Technology Project for Control and Prevention of Major Infectious Diseases in China (2018ZX10301401 to Z.Z.)China Postdoctoral Science Foundation (2020M670031 to Y.L.)
文摘The outbreak of coronavirus disease 2019(COVID-19) caused by SARS-CoV-2 has created a global health crisis. SARS-CoV-2 infects varieties of tissues where the known receptor ACE2 is low or almost absent, suggesting the existence of alternative viral entry pathways. Here, we performed a genome-wide barcoded-CRISPRa screen to identify novel host factors that enable SARS-CoV-2 infection. Beyond known host proteins, i.e., ACE2, TMPRSS2, and NRP1, we identified multiple host components,among which LDLRAD3, TMEM30A, and CLEC4G were confirmed as functional receptors for SARS-CoV-2. All these membrane proteins bind directly to spike’s N-terminal domain(NTD). Their essential and physiological roles have been confirmed in either neuron or liver cells. In particular, LDLRAD3 and CLEC4G mediate SARS-CoV-2 entry and infection in an ACE2-independent fashion. The identification of the novel receptors and entry mechanisms could advance our understanding of the multiorgan tropism of SARS-CoV-2, and may shed light on the development of COVID-19 countermeasures.
基金We are grateful to the National Natural Science Foundation of China(Grant NO.201808248)the Natural Science Foundation of Guangdong Province(NO.2018A030310255)the Project of the Scientific and Technical Program of Guangzhou(No.201607010028)for their financial support.
文摘α-Pinene is an important monoterpene,which is widely used as a flavoring agent and in fragrances,pharmaceuticals and biofuels.Although an evolved strain Escherichia coli YZFP,which had higher tolerance to pinene and titer,has been successfully used to produce high levels of pinene,the pinene titer is much lower than that of hemiterpene(isoprene)and sesquiterpenes(farnesene)to date.Moreover,the overall cellular physiological and metabolic changes caused by higher tolerance to pinene and overproduction of pinene remains unclear.To reveal the mechanism of Escherichia coli YZFP with the higher tolerance to pinene and titer,a comparative genomics and transcriptional level analyses combining with CRISPR activation(CRISPRa)and interference(CRISPRi)were carried out.The results show that the tolerance to pinene and the overproduction of pinene in E.coli may be associated with:1)the mutations of the DXP pathway genes,the rpoA and some membrane protein genes,and their upregulations of transcription levels;and 2)the mutations of some genes and their downregulation of transcriptional levels.These comparative omics analyses provided some genetic modification strategies to further improve pinene production.Overexpression of the mutated cbpA,tabA,pitA,rpoA,sufBCDS,mutS,ispH,oppF,dusB,dnaK,dxs,dxr and flgFGH genes further improved pinene production.This study also demonstrated that combining comparative omics analysis with CRISPRa and CRISPRi is an efficient technology to quickly find a new metabolic engineering strategy.
基金supported by the Biological Breeding-Major Projects (2023ZD04074)National Natural Science Foundation of China (32270433,32101205 and 32072045)+1 种基金Natural Science Foundation of Sichuan Province (2022NSFSC0143)supported by the NSF Plant Genome Research Program (IOS-2029889 and IOS-2132693)。
文摘CRISPR-Cas12a genome engineering systems have been widely used in plant research and crop breeding.To date,the performance and use of anti-CRISPR-Cas12a systems have not been fully established in plants.Here,we conduct in silico analysis to identify putative anti-CRISPR systems for Cas12a.These putative anti-CRISPR proteins,along with known anti-CRISPR proteins,are assessed for their ability to inhibit Cas12a cleavage activity in vivo and in planta.Among all anti-CRISPR proteins tested,AcrVA1 shows robust inhibition of Mb2Cas12a and LbCas12a in E.coli.Further tests show that Acr VA1 inhibits LbCas12a mediated genome editing in rice protoplasts and stable transgenic lines.Impressively,co-expression of Acr VA1 mitigates off-target effects by CRISPR-LbCas12a,as revealed by whole genome sequencing.In addition,transgenic plants expressing AcrVA1 exhibit different levels of inhibition to LbCas12a mediated genome editing,representing a novel way of fine-tuning genome editing efficiency.By controlling temporal and spatial expression of AcrVA1,we show that inducible and tissue specific genome editing can be achieved in plants.Furthermore,we demonstrate that AcrVA1 also inhibits Lb Cas12a-based CRISPR activation(CRISPRa)and based on this principle we build logic gates to turn on and off target genes in plant cells.Together,we have established an efficient anti-CRISPR-Cas12a system in plants and demonstrate its versatile applications in mitigating off-target effects,finetuning genome editing efficiency,achieving spatial-temporal control of genome editing,and generating synthetic logic gates for controlling target gene expression in plant cells.
