The most common and abundant DNA modification is 5-methylcytosine（5mC）,which has been well-established as an epigenetic mark regulating gene expression in eukaryotes（Jones,2012）.Another DNA modification N^6-methyl...The most common and abundant DNA modification is 5-methylcytosine（5mC）,which has been well-established as an epigenetic mark regulating gene expression in eukaryotes（Jones,2012）.Another DNA modification N^6-methyldeoxyadenosine（6mA）,previously reported as a widespread DNA methylation in prokaryotes.展开更多
To improve the performance of yeast cell factories for industrial production,extensive CRISPR-mediated genome editing systems have been applied by artificially creating double-strand breaks(DSBs)to introduce mutations...To improve the performance of yeast cell factories for industrial production,extensive CRISPR-mediated genome editing systems have been applied by artificially creating double-strand breaks(DSBs)to introduce mutations with the assistance of intracellular DSB repair.Diverse strategies of DSB repair are required to meet various demands,including precise editing or random editing with customized gRNAs or a gRNA library.Although most yeasts remodeling techniques have shown rewarding performance in laboratory verification,industrial yeast strain manipulation relies only on very limited strategies.Here,we comprehensively reviewed the molecular mechanisms underlying recent industrial applications to provide new insights into DSB cleavage and repair pathways in both Saccharomyces cerevisiae and other unconventional yeast species.The discussion of DSB repair covers the most frequently used homologous recombination(HR)and nonhomologous end joining(NHEJ)strategies to the less well-studied illegitimate recombination(IR)pathways,such as single-strand annealing(SSA)and microhomology-mediated end joining(MMEJ).Various CRISPR-based genome editing tools and corresponding gene editing efficiencies are described.Finally,we summarize recently developed CRISPR-based strategies that use optimized DSB repair for genome-scale editing,providing a direction for further development of yeast genome editing.展开更多
Synthetic biology is a newly developed field of research focused on designing and rebuilding novel biomolecular components, circuits, and networks. Synthetic biology can also help understand biological principles and ...Synthetic biology is a newly developed field of research focused on designing and rebuilding novel biomolecular components, circuits, and networks. Synthetic biology can also help understand biological principles and engineer complex artificial metabolic systems. DNA manipulation on a large genome-wide scale is an inevitable challenge, but a necessary tool for synthetic biology. To improve the methods used for the synthesis of long DNA fragments, here we constructed a novel shuttle vector named p GF(plasmid Genome Fast) for DNA assembly in vivo. The BAC plasmid p CC1 BAC, which can accommodate large DNA molecules, was chosen as the backbone. The sequence of the yeast artificial chromosome(YAC) regulatory element CEN6-ARS4 was synthesized and inserted into the plasmid to enable it to replicate in yeast. The selection sequence HIS3, obtained by polymerase chain reaction(PCR) from the plasmid p BS313, was inserted for screening. This new synthetic shuttle vector can mediate the transformation-associated recombination(TAR) assembly of large DNA fragments in yeast, and the assembled products can be transformed into Escherichia coli for further amplification. We also conducted in vivo DNA assembly using p GF and yeast homologous recombination and constructed a 31-kb long DNA sequence from the cyanophage PP genome. Our findings show that this novel shuttle vector would be a useful tool for efficient genome-scale DNA reconstruction.展开更多
Genomic rearrangements play a crucial role in shaping biological phenotypic diversity and driving species evolution.Synthetic chromosome rearrangement and modification by LoxP-mediated evolution(SCRaMbLE)has been appl...Genomic rearrangements play a crucial role in shaping biological phenotypic diversity and driving species evolution.Synthetic chromosome rearrangement and modification by LoxP-mediated evolution(SCRaMbLE)has been applied to explore large-scale genomic rearrangements,yet it has been observed that these rearrangements occur exclusively in genomic regions containing loxPsym sites.Here,we found that SCRaMbLE of synthetic yeast harboring synthetic chromosome V and X can generate a variety of synthetic segment insertions into wild-type chromosomes,ranging from 1 to 300 kb.Furthermore,it was revealed that the novel insertions impacted the transcriptional level of neighboring regions and affected the production of exemplar pathway of zeaxanthin.Collectively,our results improve the understanding of the ability of SCRaMbLE to generate complex structural variations in nonsynthetic regions and provide a potential model to explore genomic transposable events.展开更多
基金supported by Recruitment program of Global Youth Expert of China (to X.G.)the Elite Youth Program of the Chinese Academy of Agricultural Science (to X.G.)the intramural research support from Biotechnology Research Institute, Chinese Academy of Agricultural Sciences
文摘The most common and abundant DNA modification is 5-methylcytosine（5mC）,which has been well-established as an epigenetic mark regulating gene expression in eukaryotes（Jones,2012）.Another DNA modification N^6-methyldeoxyadenosine（6mA）,previously reported as a widespread DNA methylation in prokaryotes.
基金supported by the National Key Research and Development Program of China(No.2021YFC2101203)the General project of National Natural Science Foundation of China(No.22078021).
文摘To improve the performance of yeast cell factories for industrial production,extensive CRISPR-mediated genome editing systems have been applied by artificially creating double-strand breaks(DSBs)to introduce mutations with the assistance of intracellular DSB repair.Diverse strategies of DSB repair are required to meet various demands,including precise editing or random editing with customized gRNAs or a gRNA library.Although most yeasts remodeling techniques have shown rewarding performance in laboratory verification,industrial yeast strain manipulation relies only on very limited strategies.Here,we comprehensively reviewed the molecular mechanisms underlying recent industrial applications to provide new insights into DSB cleavage and repair pathways in both Saccharomyces cerevisiae and other unconventional yeast species.The discussion of DSB repair covers the most frequently used homologous recombination(HR)and nonhomologous end joining(NHEJ)strategies to the less well-studied illegitimate recombination(IR)pathways,such as single-strand annealing(SSA)and microhomology-mediated end joining(MMEJ).Various CRISPR-based genome editing tools and corresponding gene editing efficiencies are described.Finally,we summarize recently developed CRISPR-based strategies that use optimized DSB repair for genome-scale editing,providing a direction for further development of yeast genome editing.
基金supported by the 973 program,Grant No.2012CB721102
文摘Synthetic biology is a newly developed field of research focused on designing and rebuilding novel biomolecular components, circuits, and networks. Synthetic biology can also help understand biological principles and engineer complex artificial metabolic systems. DNA manipulation on a large genome-wide scale is an inevitable challenge, but a necessary tool for synthetic biology. To improve the methods used for the synthesis of long DNA fragments, here we constructed a novel shuttle vector named p GF(plasmid Genome Fast) for DNA assembly in vivo. The BAC plasmid p CC1 BAC, which can accommodate large DNA molecules, was chosen as the backbone. The sequence of the yeast artificial chromosome(YAC) regulatory element CEN6-ARS4 was synthesized and inserted into the plasmid to enable it to replicate in yeast. The selection sequence HIS3, obtained by polymerase chain reaction(PCR) from the plasmid p BS313, was inserted for screening. This new synthetic shuttle vector can mediate the transformation-associated recombination(TAR) assembly of large DNA fragments in yeast, and the assembled products can be transformed into Escherichia coli for further amplification. We also conducted in vivo DNA assembly using p GF and yeast homologous recombination and constructed a 31-kb long DNA sequence from the cyanophage PP genome. Our findings show that this novel shuttle vector would be a useful tool for efficient genome-scale DNA reconstruction.
基金supported by the National Key Research and Development Program of China(Grant No.2021YFC2100800)the National Natural Science Foundation of China(Grant No.22208241)+1 种基金China Postdoctoral Science Foundation(Grant No.2023M732591)the Key R&D Program of Shandong Province,China(Grant No.2022SFGC0102).
文摘Genomic rearrangements play a crucial role in shaping biological phenotypic diversity and driving species evolution.Synthetic chromosome rearrangement and modification by LoxP-mediated evolution(SCRaMbLE)has been applied to explore large-scale genomic rearrangements,yet it has been observed that these rearrangements occur exclusively in genomic regions containing loxPsym sites.Here,we found that SCRaMbLE of synthetic yeast harboring synthetic chromosome V and X can generate a variety of synthetic segment insertions into wild-type chromosomes,ranging from 1 to 300 kb.Furthermore,it was revealed that the novel insertions impacted the transcriptional level of neighboring regions and affected the production of exemplar pathway of zeaxanthin.Collectively,our results improve the understanding of the ability of SCRaMbLE to generate complex structural variations in nonsynthetic regions and provide a potential model to explore genomic transposable events.
