Gene regulation is central to all aspects of organism growth,and understanding it using large-scale functional datasets can provide a whole view of biological processes controlling complex phenotypic traits in crops.H...Gene regulation is central to all aspects of organism growth,and understanding it using large-scale functional datasets can provide a whole view of biological processes controlling complex phenotypic traits in crops.However,the connection between massive functional datasets and trait-associated gene discovery for crop improvement is still lacking.In this study,we constructed a wheat integrative gene regulatory network(wGRN)by combining an updated genome annotation and diverse complementary functional datasets,including gene expression,sequence motif,transcription factor(TF)binding,chromatin accessibility,and evolutionarily conserved regulation.wGRN contains 7.2 million genome-wide interactions covering 5947 TFs and 127439 target genes,which were further verified using known regulatory relationships,condition-specific expression,gene functional information,and experiments.We used wGRN to assign genome-wide genes to 3891 specific biological pathways and accurately prioritize candidate genes associated with complex phenotypic traits in genome-wide association studies.In addition,wGRN was used to enhance the interpretation of a spike temporal transcriptome dataset to construct high-resolution networks.We further unveiled novel regulators that enhance the power of spike phenotypic trait prediction using machine learning and contribute to the spike phenotypic differences among modern wheat accessions.Finally,we developed an interactive webserver,wGRN(http://wheat.cau.edu.cn/wGRN),for the community to explore gene regulation and discover trait-associated genes.Collectively,this community resource establishes the foundation for using large-scale functional datasets to guide trait-associated gene discovery for crop improvement.展开更多
Existing CRISPR-based genome editing techniques for Bacillus subtilis(B.subtilis)are limited due to the large size of the cas gene.IscB,a recently reported DNA nuclease,is one-third the size of Cas9,making it a potent...Existing CRISPR-based genome editing techniques for Bacillus subtilis(B.subtilis)are limited due to the large size of the cas gene.IscB,a recently reported DNA nuclease,is one-third the size of Cas9,making it a potential tool for genome editing;however,its application in B.subtilis remains unexplored.In this study,two IscB and enhanced IscB(enIscB)-based genome editing systems,named pBsuIscB and pBsuenIscB were established in B.subtilis SCK6,and their deletion efficiencies ranging from 13.3%to 100%.Compared to the pBsuIscB system,the pBsuenIscB system showed higher deletion efficiency,inducing the deletion of a large genomic fragment with a singleωRNA.Additionally,the pBsuenIscB system could integrate both single-copy and multi-copy mCherry genes in the B.subtilis SCK6 genome.Lastly,the pBsuenIscB system could efficiently conduct a second round of genome editing in B.subtilis SCK6.This study indicates that IscB can be used for genome editing in B.subtilis,enabling the efficient construction of engineered B.subtilis strains for large-scale biomolecule production.展开更多
The important methylotrophic yeast Pichia pastoris has been utilized for the production of a variety of heterologous recombinant proteins and has great potential for use in the production of value-added compounds usin...The important methylotrophic yeast Pichia pastoris has been utilized for the production of a variety of heterologous recombinant proteins and has great potential for use in the production of value-added compounds using methanol as a substrate.However,the lack of convenient and efficient genome engineering tools has hindered further applications of P.pastoris,especially in complex and multistep metabolic engineering scenarios.Hence,we developed a rapid and convenient multi-gene editing system based on CRISPR/Cas9 by optimizing the guide RNA processing strategy,which can achieve dual-gene knockout or multi-gene integration in single step.Firstly,we found that the HgH(HH-sgRNA-HDV)structure achieved the highest single-gene knockout efficiency(95.8%)among the three sgRNA processing cassettes,including a tRNA-sgRNA-tRNA(tgt)array,HgH structure and tRNA-sgRNA-HDV(tgH)structure.Furthermore,the dHgH structure(double HgH)enabled one-step dual-gene disruption and multi-gene integration.The efficiency of dual-site knockout ranged from 60%to 100%,with functional genes knockout achieving approximately 60%(Δaox1Δgut1),while dual neutral sites knockout reached 100%.Finally,we applied the system for one-step production of fatty acids and 5-hydroxytryptophan.The yield of FFAs reached 23 mg/L/μg protein/OD,while the yield of 5-hydroxytryptophan was 13.3 mg/L.The system will contribute to the application of P.pastoris as an attractive cell factory for multiplexed compound biosynthesis and will serve as a valuable tool for enhancing one-carbon(C1)bio-utilization.展开更多
基金supported by the National Key Research and Development Program of China(2021YFD1200104)the National Natural Science Foundation of China(31991210)+2 种基金the Strategic International Science and Technology Innovation Collaboration Project(2020YFE0202300)the 2115 Talent Development Program of China Agricultural Universitysupported by High-performance Computing Platform of China Agricultural University.
文摘Gene regulation is central to all aspects of organism growth,and understanding it using large-scale functional datasets can provide a whole view of biological processes controlling complex phenotypic traits in crops.However,the connection between massive functional datasets and trait-associated gene discovery for crop improvement is still lacking.In this study,we constructed a wheat integrative gene regulatory network(wGRN)by combining an updated genome annotation and diverse complementary functional datasets,including gene expression,sequence motif,transcription factor(TF)binding,chromatin accessibility,and evolutionarily conserved regulation.wGRN contains 7.2 million genome-wide interactions covering 5947 TFs and 127439 target genes,which were further verified using known regulatory relationships,condition-specific expression,gene functional information,and experiments.We used wGRN to assign genome-wide genes to 3891 specific biological pathways and accurately prioritize candidate genes associated with complex phenotypic traits in genome-wide association studies.In addition,wGRN was used to enhance the interpretation of a spike temporal transcriptome dataset to construct high-resolution networks.We further unveiled novel regulators that enhance the power of spike phenotypic trait prediction using machine learning and contribute to the spike phenotypic differences among modern wheat accessions.Finally,we developed an interactive webserver,wGRN(http://wheat.cau.edu.cn/wGRN),for the community to explore gene regulation and discover trait-associated genes.Collectively,this community resource establishes the foundation for using large-scale functional datasets to guide trait-associated gene discovery for crop improvement.
基金supported by the National Natural Science Founda-tion of China(Number:32402894)the Sichuan Science and Technology Program(Number:2024NSFSC0373).
文摘Existing CRISPR-based genome editing techniques for Bacillus subtilis(B.subtilis)are limited due to the large size of the cas gene.IscB,a recently reported DNA nuclease,is one-third the size of Cas9,making it a potential tool for genome editing;however,its application in B.subtilis remains unexplored.In this study,two IscB and enhanced IscB(enIscB)-based genome editing systems,named pBsuIscB and pBsuenIscB were established in B.subtilis SCK6,and their deletion efficiencies ranging from 13.3%to 100%.Compared to the pBsuIscB system,the pBsuenIscB system showed higher deletion efficiency,inducing the deletion of a large genomic fragment with a singleωRNA.Additionally,the pBsuenIscB system could integrate both single-copy and multi-copy mCherry genes in the B.subtilis SCK6 genome.Lastly,the pBsuenIscB system could efficiently conduct a second round of genome editing in B.subtilis SCK6.This study indicates that IscB can be used for genome editing in B.subtilis,enabling the efficient construction of engineered B.subtilis strains for large-scale biomolecule production.
基金supported by the National Key Research and Development Program of China(2021YFA0911000 and 2020YFA0907800)Key-Area Research and Development Program of Guangdong Province(2022B1111080005)+3 种基金the National Natural Science Foundation of China(NSFC 32071416)the Shenzhen Institute of Synthetic Biology Scientific Research Program(Grant No.JCHZ20200003)Shenzhen Key Laboratory for the Intelligent Microbial Manufacturing of Medicines(ZDSYS20210623091810032)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB0480000).
文摘The important methylotrophic yeast Pichia pastoris has been utilized for the production of a variety of heterologous recombinant proteins and has great potential for use in the production of value-added compounds using methanol as a substrate.However,the lack of convenient and efficient genome engineering tools has hindered further applications of P.pastoris,especially in complex and multistep metabolic engineering scenarios.Hence,we developed a rapid and convenient multi-gene editing system based on CRISPR/Cas9 by optimizing the guide RNA processing strategy,which can achieve dual-gene knockout or multi-gene integration in single step.Firstly,we found that the HgH(HH-sgRNA-HDV)structure achieved the highest single-gene knockout efficiency(95.8%)among the three sgRNA processing cassettes,including a tRNA-sgRNA-tRNA(tgt)array,HgH structure and tRNA-sgRNA-HDV(tgH)structure.Furthermore,the dHgH structure(double HgH)enabled one-step dual-gene disruption and multi-gene integration.The efficiency of dual-site knockout ranged from 60%to 100%,with functional genes knockout achieving approximately 60%(Δaox1Δgut1),while dual neutral sites knockout reached 100%.Finally,we applied the system for one-step production of fatty acids and 5-hydroxytryptophan.The yield of FFAs reached 23 mg/L/μg protein/OD,while the yield of 5-hydroxytryptophan was 13.3 mg/L.The system will contribute to the application of P.pastoris as an attractive cell factory for multiplexed compound biosynthesis and will serve as a valuable tool for enhancing one-carbon(C1)bio-utilization.
