User-friendly tools for robust transcriptional activation of endogenous genes are highly demanded in plants. We previously showed that a dCas9-VP64 system consisting of the deactivated CRISPR- associated protein 9 （d...User-friendly tools for robust transcriptional activation of endogenous genes are highly demanded in plants. We previously showed that a dCas9-VP64 system consisting of the deactivated CRISPR- associated protein 9 （dCasg） fused with four tandem repeats of the transcriptional activator VP16 0/1=64） could be used for transcriptional activation of endogenous genes in plants. In this study, we developed a second generation of vector systems for enhanced transcriptional activation in plants. We tested multiple strategies for dCasg-based transcriptional activation, and found that simultaneous recruitment of VP64 by dCas9 and a modified guide RNA scaffold gRNA2.0 （designated CRISPR-Act2.0） yielded stronger transcrip- tional activation than the dCas9-VP64 system. Moreover, we developed a multiplex transcription activator- likeeffector activation （mTALE-Act） system for simultaneous activation of up to four genes in plants. Our results suggest that mTALE-Act is even more effective than CRISPR-Act2.0 in most cases tested. In addition, we explored tissue-specific gene activation using positive feedback loops. Interestingly, our study revealed that certain endogenous genes are more amenable than others to transcriptional activation, and tightly regulated genes may cause target gene silencing when perturbed by activation probes. Hence, these new tools could be used to investigate gene regulatory networks and their control mechanisms. Assembly of multiplex CRISPR-Act2.0 and mTALE-Act systems are both based on streamlined and PCR-independent Golden Gate and Gateway cloning strategies, which will facilitate transcriptional activation applications in both dicots and monocots.展开更多
Derived from the bacterial adaptive immune system,CRISPR technology has revolutionized conventional genetic engineering methods and unprecedentedly facilitated strain engineering.In this review,we outline the fundamen...Derived from the bacterial adaptive immune system,CRISPR technology has revolutionized conventional genetic engineering methods and unprecedentedly facilitated strain engineering.In this review,we outline the fundamental CRISPR tools that have been employed for strain optimization.These tools include CRISPR editing,CRISPR interference,CRISPR activation and protein imaging.To further characterize the CRISPR technology,we present current applications of these tools in microbial systems,including model-and non-model industrial microorganisms.Specially,we point out the major challenges of the CRISPR tools when utilized for multiplex genome editing and sophisticated expression regulation.To address these challenges,we came up with strategies that place emphasis on the amelioration of DNA repair efficiency through CRISPR-Cas9-assisted recombineering.Lastly,multiple promising research directions were proposed,mainly focusing on CRISPR-based construction of microbial ecosystems toward high production of desired chemicals.展开更多
Human pluripotent stem cells （hPSCs） are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and ...Human pluripotent stem cells （hPSCs） are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR.ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline （Dox） inducible dCasg-VP64-p65-Rta （dCas9-VPR） transcription activator and a reverse Tet transactivator （rtTA） expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG pro- moter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naive pluripotsnt gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass （ICM） of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.展开更多
CRISPR-Cas9,-Cas12a,-Cas12b,and-Cas13 have been harnessed for genome engineering in human and plant cells(Liu et al.,2022).However,the large size of these Cas proteins(e.g.190 kDa for SpCas9)makes them difficult to de...CRISPR-Cas9,-Cas12a,-Cas12b,and-Cas13 have been harnessed for genome engineering in human and plant cells(Liu et al.,2022).However,the large size of these Cas proteins(e.g.190 kDa for SpCas9)makes them difficult to deliver into cells via a viral vector.The development of smaller Cas proteins will lead to reduced viral vector sizes that can be more widely adopted in versatile genome engineering systems.Recently,a CRISPR-Cas12j2(CasF)system was discovered in huge phages and developed into a hypercompact genome editor due to the small size of Cas12j2(80 kDa)(Pausch et al.,2020).Unfortunately,the gene editing efficiency of Cas12j2 in Arabidopsis protoplasts using ribonucleoprotein delivery was less than one percent(Pausch et al.,2020).Further optimization of this system is clearly required if CRISPR-Cas12j2-mediated editing in plant genomes is to be adopted by the plant sciences community.展开更多
The inducible CRISPR activation(CRISPR-a)system offers unparalleled precision and versatility for regu-lating endogenous genes,making it highly sought after in plant research.In this study,we developed a chem-ically i...The inducible CRISPR activation(CRISPR-a)system offers unparalleled precision and versatility for regu-lating endogenous genes,making it highly sought after in plant research.In this study,we developed a chem-ically inducible CRISPR-a tool for plants called ER-Tag by combining the LexA-VP16-ER inducible system with the SunTag CRISPR-a system.We systematically compared different induction strategies and achieved high efficiency in target gene activation.We demonstrated that guide RNAs can be multiplexed and pooled for large-scale screening of effective morphogenic genes and gene pairs involved in plant regeneration.Further experiments showed that induced activation of these morphogenic genes can accelerate regenera-tion and improve regeneration efficiency in both eudicot and monocot plants,including alfalfa,woodland strawberry,and sheepgrass.Our study expands the CRISPR toolset in plants and provides a powerful new strategy for studying gene function when constitutive expression is not feasible or ideal.展开更多
The clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different organisms.Current CRISPR-activation(CRISPR...The clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different organisms.Current CRISPR-activation(CRISPRa)platforms often require multiple components because of inefficient transcriptional activation.Here,we fused different phase-separation proteins to dCas9-VPR(dCas9-VP64-P65-RTA)and observed robust increases in transcriptional activation efficiency.Notably,human NUP98(nucleoporin 98)and FUS(fused in sarcoma)IDR domains were best at enhancing dCas9-VPR activity,with dCas9-VPR-FUS IDR(VPRF)outperforming the other CRISPRa systems tested in this study in both activation efficiency and system simplicity.dCas9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of dCas9-VPR.These findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.展开更多
The recently developed clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques have made it possible to reprogram target gene expression without cloning complementary DNA or disturbing geno...The recently developed clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques have made it possible to reprogram target gene expression without cloning complementary DNA or disturbing genomic sequence in mammalian cells and several multicellular organisms. We previously showed that CRISPR-associated protein 9 (Cas9) and CRISPR from Prevotella and Francisella 1 (Cpfl) could induce target mutations, deletions, inversions, and duplications both singly and multiplex in silkworm, Bombyx mori. However, it remains unknown whether the CRISPR activation (CRISPRa) system can be used in B. mori. In this study, we investigated the CRISPRa system, in which a nuclease dead Streptococcus pyogenes Cas9 (SpCas9) is fused to two transcription activation domains, including VP64 (a tetramer of the herpes simplex VP 16 transcriptional activator domain), and VPR (a tripartite activator, composed of VP64, p65, and Rta). The results showed that both dCas9-VP64 and dCas9-VPR systems could be used in B. mori cells, of which the latter showed significantly higher activity. The dCas9-VPR system showed considerable activity on all five tested target genes, and further analysis revealed that the up-regulation of genes was negatively correlated to their basal expression level. We also observed that this system could be used to upregulate a range of target genes. Taken together, our findings demonstrate that CRISPRa can be a powerful tool to study gene functions in B. mori and perhaps other non-drosophila insects.展开更多
The genus Populus has long been used for environmental,agroforestry and industrial applications worldwide.Today Populus is also recognized as a desirable crop for biofuel production and a model tree for physiological ...The genus Populus has long been used for environmental,agroforestry and industrial applications worldwide.Today Populus is also recognized as a desirable crop for biofuel production and a model tree for physiological and ecological research.As such,various modern biotechnologies,including CRISPR/Cas9-based techniques,have been actively applied to Populus for genetic and genomic improvements for traits such as increased growth rate and tailored lignin composition.However,CRISPR/Cas9 has been primarily used as the active Cas9 form to create knockouts in the hybrid poplar clone“717-1B4”(P.tremula x P.alba clone INRA 717-1B4).Alternative CRISPR/Cas9-based technologies,e.g.those involving modified Cas9 for gene activation and base editing,have not been evaluated in most Populus species for their efficacy.Here we employed a deactivated Cas9(dCas9)-based CRISPR activation(CRISPRa)technique to fine-tune the expression of two target genes,TPX2 and LecRLK-G which play important roles in plant growth and defense response,in hybrid poplar clone“717-1B4”and poplar clone“WV94”(P.deltoides“WV94”),respectively.We observed that CRISPRa resulted in 1.2-fold to 7.0-fold increase in target gene expression through transient expression in protoplasts and Agrobacterium-mediated stable transformation,demonstrating the effectiveness of dCas9-based CRISPRa system in Populus.In addition,we applied Cas9 nickase(nCas9)-based cytosine base editor(CBE)to precisely introduce premature stop codons via C-to-T conversion,with an efficiency of 13%–14%,in the target gene PLATZ which encodes a transcription factor involved in plant fungal pathogen response in hybrid poplar clone“717-1B4”.Overall,we showcase the successful application of CRISPR/Cas-based technologies in gene expression regulation and precise gene engineering in two Populus species,facilitating the adoption of emerging genome editing tools in woody species.展开更多
基金This work was supported by startup funds from East Carolina University and University of Maryland-College Park and a Collaborative Funding grant from North Carolina Biotechnology Center and Syngenta Biotechnology （2016-CFG-8003） to Y.Q. This work was also supported by grants, including the Sichuan Youth Science and Technology Foundation （2017JQ0005）, the National Science Foundation of China （31771486）, and the Fundamental Research Funds for the Central Universities （ZYGX2016J119） to Y.Z.
文摘User-friendly tools for robust transcriptional activation of endogenous genes are highly demanded in plants. We previously showed that a dCas9-VP64 system consisting of the deactivated CRISPR- associated protein 9 （dCasg） fused with four tandem repeats of the transcriptional activator VP16 0/1=64） could be used for transcriptional activation of endogenous genes in plants. In this study, we developed a second generation of vector systems for enhanced transcriptional activation in plants. We tested multiple strategies for dCasg-based transcriptional activation, and found that simultaneous recruitment of VP64 by dCas9 and a modified guide RNA scaffold gRNA2.0 （designated CRISPR-Act2.0） yielded stronger transcrip- tional activation than the dCas9-VP64 system. Moreover, we developed a multiplex transcription activator- likeeffector activation （mTALE-Act） system for simultaneous activation of up to four genes in plants. Our results suggest that mTALE-Act is even more effective than CRISPR-Act2.0 in most cases tested. In addition, we explored tissue-specific gene activation using positive feedback loops. Interestingly, our study revealed that certain endogenous genes are more amenable than others to transcriptional activation, and tightly regulated genes may cause target gene silencing when perturbed by activation probes. Hence, these new tools could be used to investigate gene regulatory networks and their control mechanisms. Assembly of multiplex CRISPR-Act2.0 and mTALE-Act systems are both based on streamlined and PCR-independent Golden Gate and Gateway cloning strategies, which will facilitate transcriptional activation applications in both dicots and monocots.
基金This work was supported by grants from National Natural Science Foundation of China(No.21276014,21476011)National High Technology Research and Development Program(863 Program)(No.2015AA021003)+1 种基金Fundamental Research Funds for the Central Universities(YS1407)111 project(B13005).
文摘Derived from the bacterial adaptive immune system,CRISPR technology has revolutionized conventional genetic engineering methods and unprecedentedly facilitated strain engineering.In this review,we outline the fundamental CRISPR tools that have been employed for strain optimization.These tools include CRISPR editing,CRISPR interference,CRISPR activation and protein imaging.To further characterize the CRISPR technology,we present current applications of these tools in microbial systems,including model-and non-model industrial microorganisms.Specially,we point out the major challenges of the CRISPR tools when utilized for multiplex genome editing and sophisticated expression regulation.To address these challenges,we came up with strategies that place emphasis on the amelioration of DNA repair efficiency through CRISPR-Cas9-assisted recombineering.Lastly,multiple promising research directions were proposed,mainly focusing on CRISPR-based construction of microbial ecosystems toward high production of desired chemicals.
文摘Human pluripotent stem cells （hPSCs） are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR.ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline （Dox） inducible dCasg-VP64-p65-Rta （dCas9-VPR） transcription activator and a reverse Tet transactivator （rtTA） expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG pro- moter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naive pluripotsnt gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass （ICM） of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.
基金supported by the National Key Research and Development Program of China(award no.NK2022010204)to Y.Z.the National Natural Science Foundation of China(award nos.32270433,32101205,32072045,and 31960423)to X.T.,X.Z.,and Y.Z.+3 种基金the Sichuan Science and Technology Program(award no.2021JDRC0032)to Y.Z.the Technology Innovation and Application Development Program of Chongqing(award no.CSTC2021JSCX-CYLHX0001)to X.T.and Y.Z.supported by the National Science Foundation Plant Genome Research Program grant(award nos.IOS-1758745 and IOS2029889)USDA-AFRI Agricultural Innovations Through Gene Editing Program(award no.2021-67013-34554)to Y.Q.S.S.is a fellow of the Foundation for Food and Agriculture Research.
文摘CRISPR-Cas9,-Cas12a,-Cas12b,and-Cas13 have been harnessed for genome engineering in human and plant cells(Liu et al.,2022).However,the large size of these Cas proteins(e.g.190 kDa for SpCas9)makes them difficult to deliver into cells via a viral vector.The development of smaller Cas proteins will lead to reduced viral vector sizes that can be more widely adopted in versatile genome engineering systems.Recently,a CRISPR-Cas12j2(CasF)system was discovered in huge phages and developed into a hypercompact genome editor due to the small size of Cas12j2(80 kDa)(Pausch et al.,2020).Unfortunately,the gene editing efficiency of Cas12j2 in Arabidopsis protoplasts using ribonucleoprotein delivery was less than one percent(Pausch et al.,2020).Further optimization of this system is clearly required if CRISPR-Cas12j2-mediated editing in plant genomes is to be adopted by the plant sciences community.
基金supported by the National Key R&D Program of China (2022YFD1500503 to X.S.)the Key Projects in Science and Technology of Inner Mongolia (2021ZD0031 to X.C.and S.Z.)+6 种基金the National Key Research and Development Program (2022YFF1002802 to X.D.)the Youth Innovation Promotion Association,CAS (Y2022039 to X.D.)the Youth Innovation Promotion Association,CAS (2022096 to S.Z.)the National Science Foundation of China (NSFC32272692 to J.Z.)Project ZR2022QC054 of the Shandong Provincial Natural Science Foundation (to Y.T.)funding from the State Key Laboratory of Protein and Plant Gene Research (to Q.L.).
文摘The inducible CRISPR activation(CRISPR-a)system offers unparalleled precision and versatility for regu-lating endogenous genes,making it highly sought after in plant research.In this study,we developed a chem-ically inducible CRISPR-a tool for plants called ER-Tag by combining the LexA-VP16-ER inducible system with the SunTag CRISPR-a system.We systematically compared different induction strategies and achieved high efficiency in target gene activation.We demonstrated that guide RNAs can be multiplexed and pooled for large-scale screening of effective morphogenic genes and gene pairs involved in plant regeneration.Further experiments showed that induced activation of these morphogenic genes can accelerate regenera-tion and improve regeneration efficiency in both eudicot and monocot plants,including alfalfa,woodland strawberry,and sheepgrass.Our study expands the CRISPR toolset in plants and provides a powerful new strategy for studying gene function when constitutive expression is not feasible or ideal.
基金supported by the National Key Research and Development Program of China(2017YFA0102801)the National Natural Science Foundation of China(91640119,81330055,and 32001063)+2 种基金the Guangdong Special Support Program(2019BT02Y276)the Natural Science Foundation of Guangdong Province(2023A1515010176)the Guangzhou Science and Technology Program key projects(2023A04J1952)。
文摘The clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different organisms.Current CRISPR-activation(CRISPRa)platforms often require multiple components because of inefficient transcriptional activation.Here,we fused different phase-separation proteins to dCas9-VPR(dCas9-VP64-P65-RTA)and observed robust increases in transcriptional activation efficiency.Notably,human NUP98(nucleoporin 98)and FUS(fused in sarcoma)IDR domains were best at enhancing dCas9-VPR activity,with dCas9-VPR-FUS IDR(VPRF)outperforming the other CRISPRa systems tested in this study in both activation efficiency and system simplicity.dCas9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of dCas9-VPR.These findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.
基金the National Natural Science Foundation of China (No.31530071)the Chongqing Postdoctoral Science Foundation (No. Xm2016030)Chongqing Research program of basic Research and Frontier Technology (No.cstc2017jcyjAX0349).
文摘The recently developed clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques have made it possible to reprogram target gene expression without cloning complementary DNA or disturbing genomic sequence in mammalian cells and several multicellular organisms. We previously showed that CRISPR-associated protein 9 (Cas9) and CRISPR from Prevotella and Francisella 1 (Cpfl) could induce target mutations, deletions, inversions, and duplications both singly and multiplex in silkworm, Bombyx mori. However, it remains unknown whether the CRISPR activation (CRISPRa) system can be used in B. mori. In this study, we investigated the CRISPRa system, in which a nuclease dead Streptococcus pyogenes Cas9 (SpCas9) is fused to two transcription activation domains, including VP64 (a tetramer of the herpes simplex VP 16 transcriptional activator domain), and VPR (a tripartite activator, composed of VP64, p65, and Rta). The results showed that both dCas9-VP64 and dCas9-VPR systems could be used in B. mori cells, of which the latter showed significantly higher activity. The dCas9-VPR system showed considerable activity on all five tested target genes, and further analysis revealed that the up-regulation of genes was negatively correlated to their basal expression level. We also observed that this system could be used to upregulate a range of target genes. Taken together, our findings demonstrate that CRISPRa can be a powerful tool to study gene functions in B. mori and perhaps other non-drosophila insects.
文摘The genus Populus has long been used for environmental,agroforestry and industrial applications worldwide.Today Populus is also recognized as a desirable crop for biofuel production and a model tree for physiological and ecological research.As such,various modern biotechnologies,including CRISPR/Cas9-based techniques,have been actively applied to Populus for genetic and genomic improvements for traits such as increased growth rate and tailored lignin composition.However,CRISPR/Cas9 has been primarily used as the active Cas9 form to create knockouts in the hybrid poplar clone“717-1B4”(P.tremula x P.alba clone INRA 717-1B4).Alternative CRISPR/Cas9-based technologies,e.g.those involving modified Cas9 for gene activation and base editing,have not been evaluated in most Populus species for their efficacy.Here we employed a deactivated Cas9(dCas9)-based CRISPR activation(CRISPRa)technique to fine-tune the expression of two target genes,TPX2 and LecRLK-G which play important roles in plant growth and defense response,in hybrid poplar clone“717-1B4”and poplar clone“WV94”(P.deltoides“WV94”),respectively.We observed that CRISPRa resulted in 1.2-fold to 7.0-fold increase in target gene expression through transient expression in protoplasts and Agrobacterium-mediated stable transformation,demonstrating the effectiveness of dCas9-based CRISPRa system in Populus.In addition,we applied Cas9 nickase(nCas9)-based cytosine base editor(CBE)to precisely introduce premature stop codons via C-to-T conversion,with an efficiency of 13%–14%,in the target gene PLATZ which encodes a transcription factor involved in plant fungal pathogen response in hybrid poplar clone“717-1B4”.Overall,we showcase the successful application of CRISPR/Cas-based technologies in gene expression regulation and precise gene engineering in two Populus species,facilitating the adoption of emerging genome editing tools in woody species.
