Genetic lineage tracing has been widely employed to investigate cell lineages and fate.However,conventional reporting systems often label the entire cytoplasm,making it challenging to discern cell boundaries.Additiona...Genetic lineage tracing has been widely employed to investigate cell lineages and fate.However,conventional reporting systems often label the entire cytoplasm,making it challenging to discern cell boundaries.Additionally,single Cre-lox P recombination systems have limitations in tracing specific cell populations.This study proposes three reporting systems utilizing Cre,Dre,and Dre+Cre mediated recombination.These systems incorporate td Tomato expression on the cell membrane and Phi YFP expression within the nucleus,allowing for clear observation of the nucleus and membrane.The efficacy of these systems is successfully demonstrated by labeling cardiomyocytes and hepatocytes.The potential for dynamic visualization of the cell membrane is showcased using intravital imaging microscopy or threedimensional imaging.Furthermore,by combining this dual recombinase system with the Pro Tracer system,hepatocyte proliferation is traced with enhanced precision.This reporting system holds significant importance for advancing the understanding of cell fate studies in development,homeostasis,and diseases.展开更多
Genome editing is considered as the most widely used approach of the present era. It had become a basic need of the current micro and molecular biological experiments. Gene engineering finds its widespread application...Genome editing is considered as the most widely used approach of the present era. It had become a basic need of the current micro and molecular biological experiments. Gene engineering finds its widespread applications in medical, industry and agricultural sector. Unlike previous genetic engineering practices to insert or delete a part of genetic material at random place, genome editing allows the precise manipulation of DNA at a specific location. Zinc Finger Nucleases (ZFNs), Transcription Activator like Effector Nucleases (TALENs), Clustered Regularly Interspresed Short Palindromic repeats (CRISPR/Cas system) and meganucleases (recombinases) are the prime tools for editing an organism’s genome. Genome editing tools have an advantage to selectively delete or to integrate specific genes at specific loci. Use of recombinases for specifying site has further reduced time to integrate genes site specifically. Site specific gene stacking by the use of recombinases coupled with ZFNs, TALENs, or CRISPR/Cas genes have paved new pathways to target genes site specifically and to improve germplasm in lesser time than conventional breeding approaches.展开更多
Genomic integration of genes and pathway-sized DNA cassettes is often an indispensable way to construct robust and productive microbial cell factories.For some uncommon microbial hosts,such as Mycolicibacterium and My...Genomic integration of genes and pathway-sized DNA cassettes is often an indispensable way to construct robust and productive microbial cell factories.For some uncommon microbial hosts,such as Mycolicibacterium and Mycobacterium species,however,it is a challenge.Here,we present a multiplexed integrase-assisted site-specific recombination(miSSR)method to precisely and iteratively integrate genes/pathways with controllable copies in the chromosomes of Mycolicibacteria for the purpose of developing cell factories.First,a single-step multi-copy integration method was established in M.neoaurum by a combination application of mycobacteriophage L5 integrase and two-step allelic exchange strategy,the efficiencies of which were~100%for no more than three-copy integration events and decreased sharply to~20%for five-copy integration events.Second,the R4,Bxb1 andΦC31 bacteriophage Att/Int systems were selected to extend the available integration toolbox for multiplexed gene integration events.Third,a reconstructed mycolicibacterial Xer recombinases(Xer-cise)system was employed to recycle the selection marker of gene recombination to facilitate the iterative gene manipulation.As a proof of concept,the biosynthetic pathway of ergothioneine(EGT)in Mycolicibacterium neoaurum ATCC 25795 was achieved by remodeling its metabolic pathway with a miSSR system.With six copies of the biosynthetic gene clusters(BGCs)of EGT and pentose phosphate isomerase(PRT),the titer of EGT in the resulting strain in a 30 mL shake flask within 5 days was enhanced to 66 mg/L,which was 3.77 times of that in the wild strain.The improvements indicated that the miSSR system was an effective,flexible,and convenient tool to engineer the genomes of Mycolicibacteria as well as other strains in the Mycobacteriaceae due to their proximate evolutionary relationships.展开更多
基金supported by the National Key Research&Development Program of China(2021YFA0805100,2023YFA1800700,2019YFA0110403,2019YFA0802000)the National Science Foundation of China(82088101,32370885,92368103,32370897)the Westlake Education Foundation,and the Benyuan Charity Fund,Research Funds of Hangzhou Institute for Advanced Study(2022ZZ01015 and B04006C01600515)。
文摘Genetic lineage tracing has been widely employed to investigate cell lineages and fate.However,conventional reporting systems often label the entire cytoplasm,making it challenging to discern cell boundaries.Additionally,single Cre-lox P recombination systems have limitations in tracing specific cell populations.This study proposes three reporting systems utilizing Cre,Dre,and Dre+Cre mediated recombination.These systems incorporate td Tomato expression on the cell membrane and Phi YFP expression within the nucleus,allowing for clear observation of the nucleus and membrane.The efficacy of these systems is successfully demonstrated by labeling cardiomyocytes and hepatocytes.The potential for dynamic visualization of the cell membrane is showcased using intravital imaging microscopy or threedimensional imaging.Furthermore,by combining this dual recombinase system with the Pro Tracer system,hepatocyte proliferation is traced with enhanced precision.This reporting system holds significant importance for advancing the understanding of cell fate studies in development,homeostasis,and diseases.
文摘Genome editing is considered as the most widely used approach of the present era. It had become a basic need of the current micro and molecular biological experiments. Gene engineering finds its widespread applications in medical, industry and agricultural sector. Unlike previous genetic engineering practices to insert or delete a part of genetic material at random place, genome editing allows the precise manipulation of DNA at a specific location. Zinc Finger Nucleases (ZFNs), Transcription Activator like Effector Nucleases (TALENs), Clustered Regularly Interspresed Short Palindromic repeats (CRISPR/Cas system) and meganucleases (recombinases) are the prime tools for editing an organism’s genome. Genome editing tools have an advantage to selectively delete or to integrate specific genes at specific loci. Use of recombinases for specifying site has further reduced time to integrate genes site specifically. Site specific gene stacking by the use of recombinases coupled with ZFNs, TALENs, or CRISPR/Cas genes have paved new pathways to target genes site specifically and to improve germplasm in lesser time than conventional breeding approaches.
基金supported by the National Natural Science Foundation of China(No.21776075)the Natural Science Foundation of Shanghai(No.20ZR1415100)the National Key Research and Development Program of China(No.SQ2020YFC210061).
文摘Genomic integration of genes and pathway-sized DNA cassettes is often an indispensable way to construct robust and productive microbial cell factories.For some uncommon microbial hosts,such as Mycolicibacterium and Mycobacterium species,however,it is a challenge.Here,we present a multiplexed integrase-assisted site-specific recombination(miSSR)method to precisely and iteratively integrate genes/pathways with controllable copies in the chromosomes of Mycolicibacteria for the purpose of developing cell factories.First,a single-step multi-copy integration method was established in M.neoaurum by a combination application of mycobacteriophage L5 integrase and two-step allelic exchange strategy,the efficiencies of which were~100%for no more than three-copy integration events and decreased sharply to~20%for five-copy integration events.Second,the R4,Bxb1 andΦC31 bacteriophage Att/Int systems were selected to extend the available integration toolbox for multiplexed gene integration events.Third,a reconstructed mycolicibacterial Xer recombinases(Xer-cise)system was employed to recycle the selection marker of gene recombination to facilitate the iterative gene manipulation.As a proof of concept,the biosynthetic pathway of ergothioneine(EGT)in Mycolicibacterium neoaurum ATCC 25795 was achieved by remodeling its metabolic pathway with a miSSR system.With six copies of the biosynthetic gene clusters(BGCs)of EGT and pentose phosphate isomerase(PRT),the titer of EGT in the resulting strain in a 30 mL shake flask within 5 days was enhanced to 66 mg/L,which was 3.77 times of that in the wild strain.The improvements indicated that the miSSR system was an effective,flexible,and convenient tool to engineer the genomes of Mycolicibacteria as well as other strains in the Mycobacteriaceae due to their proximate evolutionary relationships.
