Transformation-associated recombination(TAR)has been widely used to assemble large DNA constructs.One of the significant obstacles hindering assembly efficiency is the presence of error-prone DNA repair pathways in ye...Transformation-associated recombination(TAR)has been widely used to assemble large DNA constructs.One of the significant obstacles hindering assembly efficiency is the presence of error-prone DNA repair pathways in yeast,which results in vector backbone recircularization or illegitimate recombination products.To increase TAR assembly efficiency,we prepared a dual-selective TAR vector,pGFCS,by adding a PADH1-URA3 cassette to a previously described yeast-bacteria shuttle vector,p GF,harboring a PHIS3–HIS3 cassette as a positive selection marker.This new cassette works as a negative selection marker to ensure that yeast harboring a recircularized vector cannot propagate in the presence of 5-fluoroorotic acid.To prevent pGFCS bearing ura3 from recombining with endogenous ura3-52 in the yeast genome,a highly transformable Saccharomyces cerevisiae strain,VL6-48B,was prepared by chromosomal substitution of ura3-52 with a transgene conferring resistance to blasticidin.A55-kb genomic fragment of monkeypox virus encompassing primary detection targets for quantitative PCR was assembled by TAR using pGFCS in VL6-48B.The pGFCS-mediated TAR assembly showed a zero rate of vector recircularization and an average correct assembly yield of 79%indicating that the dual-selection strategy provides an efficient approach to optimizing TAR assembly.展开更多
Viral infectious clones(ICs)serve as robust platforms for studying viral biology and screening antiviral agents using reverse genetics.However,the molecular profiles and complex limitations of human coronaviruses(HCoV...Viral infectious clones(ICs)serve as robust platforms for studying viral biology and screening antiviral agents using reverse genetics.However,the molecular profiles and complex limitations of human coronaviruses(HCoVs)pose a challenge to ICs development.In this study,we report a novel platform to develop the ICs for HCoV-OC43-VR1558 using a one-step assembly method in yeast by transformation-associated recombination(TAR)technology.Recombinant HCoV-OC43-VR1558,named as rOC43(1558)-WT,was rapidly generated by TAR.In addition,recombinant HCoV-OC43-VR1558-expressing reporter genes,named as rOC43(1558)-ns2FusionRluc,was also generated based on TAR by inserting the ns2 region of the IC with Renilla luciferase(Rluc).We further characterized their replication through virus titration using 50%tissue culture infective dose(TCID50)and indirect immunofluorescence assay(IFA),luciferase reporter assay,and western blotting(WB)assay.The genetic stability of the recombinant HCoV-OC43 was assessed through viral genome sequencing following passaging in BHK-21 cells.These reporter viruses were validated as screening tools for inhibitorsin vitro by evaluating the antiviral activities of remdesivir and chloroquine.The phenotypes of HCoV-OC43-VR1558 and HCoV-OC43-VR759 were comparedin vitro andin vivo.The TAR-based one-step assembly of IC was successfully applied,facilitating the rapid generation of recombinant HCoV-OC43 and providing a useful platform for the investigation of biological mechanisms,development of vaccines and diagnostic tests,and screening inhibitors of HCoVs.展开更多
基金supported by the National Science and Technology Major Project of China(2018ZX10711001-006)the Key Research Program of the Chinese Academy of Sciences(KJZD-SW-L06-02)。
文摘Transformation-associated recombination(TAR)has been widely used to assemble large DNA constructs.One of the significant obstacles hindering assembly efficiency is the presence of error-prone DNA repair pathways in yeast,which results in vector backbone recircularization or illegitimate recombination products.To increase TAR assembly efficiency,we prepared a dual-selective TAR vector,pGFCS,by adding a PADH1-URA3 cassette to a previously described yeast-bacteria shuttle vector,p GF,harboring a PHIS3–HIS3 cassette as a positive selection marker.This new cassette works as a negative selection marker to ensure that yeast harboring a recircularized vector cannot propagate in the presence of 5-fluoroorotic acid.To prevent pGFCS bearing ura3 from recombining with endogenous ura3-52 in the yeast genome,a highly transformable Saccharomyces cerevisiae strain,VL6-48B,was prepared by chromosomal substitution of ura3-52 with a transgene conferring resistance to blasticidin.A55-kb genomic fragment of monkeypox virus encompassing primary detection targets for quantitative PCR was assembled by TAR using pGFCS in VL6-48B.The pGFCS-mediated TAR assembly showed a zero rate of vector recircularization and an average correct assembly yield of 79%indicating that the dual-selection strategy provides an efficient approach to optimizing TAR assembly.
基金supported by the National Key Research and Development Program of China(2022YFC2304100 and 2021YFA1201003).
文摘Viral infectious clones(ICs)serve as robust platforms for studying viral biology and screening antiviral agents using reverse genetics.However,the molecular profiles and complex limitations of human coronaviruses(HCoVs)pose a challenge to ICs development.In this study,we report a novel platform to develop the ICs for HCoV-OC43-VR1558 using a one-step assembly method in yeast by transformation-associated recombination(TAR)technology.Recombinant HCoV-OC43-VR1558,named as rOC43(1558)-WT,was rapidly generated by TAR.In addition,recombinant HCoV-OC43-VR1558-expressing reporter genes,named as rOC43(1558)-ns2FusionRluc,was also generated based on TAR by inserting the ns2 region of the IC with Renilla luciferase(Rluc).We further characterized their replication through virus titration using 50%tissue culture infective dose(TCID50)and indirect immunofluorescence assay(IFA),luciferase reporter assay,and western blotting(WB)assay.The genetic stability of the recombinant HCoV-OC43 was assessed through viral genome sequencing following passaging in BHK-21 cells.These reporter viruses were validated as screening tools for inhibitorsin vitro by evaluating the antiviral activities of remdesivir and chloroquine.The phenotypes of HCoV-OC43-VR1558 and HCoV-OC43-VR759 were comparedin vitro andin vivo.The TAR-based one-step assembly of IC was successfully applied,facilitating the rapid generation of recombinant HCoV-OC43 and providing a useful platform for the investigation of biological mechanisms,development of vaccines and diagnostic tests,and screening inhibitors of HCoVs.
