Coupling ssDNA recombineering with CRISPR-Cas9 for Escherichia coli DnaG mutations
Homologous recombination-based recombineering is a widely used DNA cloning and modification technique; recombineering efficiency improvement would be helpful for high-throughput DNA manipulation. Escherichia coli primase DnaG variants, such as DnaG Q576A and DnaG K580A, increase the recombineering e...
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| Veröffentlicht in: | Applied microbiology and biotechnology 2019-04, Vol.103 (8), p.3559-3570 |
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| creator | Li, Jing Sun, Jian Gao, Xinyue Wu, Zhixin Shang, Guangdong |
| description | Homologous recombination-based recombineering is a widely used DNA cloning and modification technique; recombineering efficiency improvement would be helpful for high-throughput DNA manipulation.
Escherichia coli
primase DnaG variants, such as DnaG Q576A and DnaG K580A, increase the recombineering efficiency via impairment of the interaction between primase and the replisome and boost the loading of more ssDNA on the replication fork. Bacterial adaptive immunity origin CRISPR-Cas9 is emerging as a powerful genome editing strategy. In this study, ssDNA recombineering and CRISPR-Cas9 were combined for the generation of DnaG variants. The tightly regulated Red operon expression cassette and tightly regulated Cas9 expression cassette were integrated into one chloroamphenicol resistance, p15A replicon-based vector. A self-curing, kanamycin resistance, p15A replicon-based plasmid was applied for the plasmid elimination after genome editing. The genome editing efficiency was as high as 100%. The recombineering efficiency of the strains harboring the DnaG variants was assayed via the kanamycin resistance gene repair as well as the chromosomal gene deletion experiments. The established genome editing strategy will expedite the DnaG structure and function relationship study as well as the metabolic engineering and synthetic biology applications. |
| doi_str_mv | 10.1007/s00253-019-09744-9 |
| format | Article |
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Escherichia coli
primase DnaG variants, such as DnaG Q576A and DnaG K580A, increase the recombineering efficiency via impairment of the interaction between primase and the replisome and boost the loading of more ssDNA on the replication fork. Bacterial adaptive immunity origin CRISPR-Cas9 is emerging as a powerful genome editing strategy. In this study, ssDNA recombineering and CRISPR-Cas9 were combined for the generation of DnaG variants. The tightly regulated Red operon expression cassette and tightly regulated Cas9 expression cassette were integrated into one chloroamphenicol resistance, p15A replicon-based vector. A self-curing, kanamycin resistance, p15A replicon-based plasmid was applied for the plasmid elimination after genome editing. The genome editing efficiency was as high as 100%. The recombineering efficiency of the strains harboring the DnaG variants was assayed via the kanamycin resistance gene repair as well as the chromosomal gene deletion experiments. The established genome editing strategy will expedite the DnaG structure and function relationship study as well as the metabolic engineering and synthetic biology applications.</description><identifier>ISSN: 0175-7598</identifier><identifier>EISSN: 1432-0614</identifier><identifier>DOI: 10.1007/s00253-019-09744-9</identifier><identifier>PMID: 30879090</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer Berlin Heidelberg</publisher><subject>Adaptive immunity ; Antibacterial agents ; Bacteria ; Biological products ; Biomedical and Life Sciences ; Biotechnology ; Chromosome deletion ; Chromosomes ; Clonal deletion ; Cloning ; CRISPR ; CRISPR-Cas Systems ; Deoxyribonucleic acid ; DNA ; DNA primase ; DNA Primase - genetics ; DNA Primase - metabolism ; DNA, Bacterial - genetics ; DNA, Single-Stranded - genetics ; E coli ; Editing ; Efficiency ; Escherichia coli ; Escherichia coli - enzymology ; Escherichia coli - genetics ; Escherichia coli Proteins - genetics ; Escherichia coli Proteins - metabolism ; Gene deletion ; Gene Editing ; Gene mutation ; Genes ; Genetic aspects ; Genetic Engineering - methods ; Genetic research ; Genome editing ; Genome, Bacterial - genetics ; Genomes ; Genomics ; Homologous Recombination ; Homology ; Immunity ; Immunotherapy ; Kanamycin ; Life Sciences ; Metabolic engineering ; Methods ; Methods and Protocols ; Microbial Genetics and Genomics ; Microbiology ; Mutation ; Neomycin ; Plasmids - genetics ; Plasmids - metabolism ; Primase ; Structure-function relationships</subject><ispartof>Applied microbiology and biotechnology, 2019-04, Vol.103 (8), p.3559-3570</ispartof><rights>Springer-Verlag GmbH Germany, part of Springer Nature 2019</rights><rights>COPYRIGHT 2019 Springer</rights><rights>Applied Microbiology and Biotechnology is a copyright of Springer, (2019). All Rights Reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c513t-c33b52dc9ab80dbe320c09baa005e61810ea723895d41b1d6f6c28860824238a3</citedby><cites>FETCH-LOGICAL-c513t-c33b52dc9ab80dbe320c09baa005e61810ea723895d41b1d6f6c28860824238a3</cites><orcidid>0000-0001-6142-3785</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00253-019-09744-9$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00253-019-09744-9$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27922,27923,41486,42555,51317</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/30879090$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Li, Jing</creatorcontrib><creatorcontrib>Sun, Jian</creatorcontrib><creatorcontrib>Gao, Xinyue</creatorcontrib><creatorcontrib>Wu, Zhixin</creatorcontrib><creatorcontrib>Shang, Guangdong</creatorcontrib><title>Coupling ssDNA recombineering with CRISPR-Cas9 for Escherichia coli DnaG mutations</title><title>Applied microbiology and biotechnology</title><addtitle>Appl Microbiol Biotechnol</addtitle><addtitle>Appl Microbiol Biotechnol</addtitle><description>Homologous recombination-based recombineering is a widely used DNA cloning and modification technique; recombineering efficiency improvement would be helpful for high-throughput DNA manipulation.
Escherichia coli
primase DnaG variants, such as DnaG Q576A and DnaG K580A, increase the recombineering efficiency via impairment of the interaction between primase and the replisome and boost the loading of more ssDNA on the replication fork. Bacterial adaptive immunity origin CRISPR-Cas9 is emerging as a powerful genome editing strategy. In this study, ssDNA recombineering and CRISPR-Cas9 were combined for the generation of DnaG variants. The tightly regulated Red operon expression cassette and tightly regulated Cas9 expression cassette were integrated into one chloroamphenicol resistance, p15A replicon-based vector. A self-curing, kanamycin resistance, p15A replicon-based plasmid was applied for the plasmid elimination after genome editing. The genome editing efficiency was as high as 100%. The recombineering efficiency of the strains harboring the DnaG variants was assayed via the kanamycin resistance gene repair as well as the chromosomal gene deletion experiments. The established genome editing strategy will expedite the DnaG structure and function relationship study as well as the metabolic engineering and synthetic biology applications.</description><subject>Adaptive immunity</subject><subject>Antibacterial agents</subject><subject>Bacteria</subject><subject>Biological products</subject><subject>Biomedical and Life Sciences</subject><subject>Biotechnology</subject><subject>Chromosome deletion</subject><subject>Chromosomes</subject><subject>Clonal deletion</subject><subject>Cloning</subject><subject>CRISPR</subject><subject>CRISPR-Cas Systems</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA primase</subject><subject>DNA Primase - genetics</subject><subject>DNA Primase - metabolism</subject><subject>DNA, Bacterial - genetics</subject><subject>DNA, Single-Stranded - genetics</subject><subject>E coli</subject><subject>Editing</subject><subject>Efficiency</subject><subject>Escherichia coli</subject><subject>Escherichia coli - enzymology</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli Proteins - genetics</subject><subject>Escherichia coli Proteins - metabolism</subject><subject>Gene deletion</subject><subject>Gene Editing</subject><subject>Gene mutation</subject><subject>Genes</subject><subject>Genetic aspects</subject><subject>Genetic Engineering - methods</subject><subject>Genetic research</subject><subject>Genome editing</subject><subject>Genome, Bacterial - genetics</subject><subject>Genomes</subject><subject>Genomics</subject><subject>Homologous Recombination</subject><subject>Homology</subject><subject>Immunity</subject><subject>Immunotherapy</subject><subject>Kanamycin</subject><subject>Life Sciences</subject><subject>Metabolic engineering</subject><subject>Methods</subject><subject>Methods and Protocols</subject><subject>Microbial Genetics and Genomics</subject><subject>Microbiology</subject><subject>Mutation</subject><subject>Neomycin</subject><subject>Plasmids - genetics</subject><subject>Plasmids - metabolism</subject><subject>Primase</subject><subject>Structure-function relationships</subject><issn>0175-7598</issn><issn>1432-0614</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNp9kc1u1DAUhS1ERYfCC7BAkdi0C5drO3Hs5Sj9YaSqRVNYW47jzLhK4sFOBLw9DlNaDULIC0vnfufoXh2E3hE4JwDlxwhAC4aBSAyyzHMsX6AFyRnFwEn-Ei2AlAUuCymO0esYHwAIFZy_QscMRClBwgKtKz_tOjdsshgvbpdZsMb3tRusDbP43Y3brFqv7j-vcaWjzFofsstotmlstk5nxncuuxj0ddZPox6dH-IbdNTqLtq3j_8J-np1-aX6hG_urlfV8gabgrARG8bqgjZG6lpAU1tGwYCstQYoLCeCgNUlZUIWTU5q0vCWGyoEB0HzJGt2gk73ubvgv002jqp30diu04P1U1SUSMaBEWAJ_fAX-uCnMKTtZorIHDiTz9RGd1a5ofVj0GYOVctCEJYzQees839Q6TW2d8YPtnVJPzCcHRgSM9of40ZPMarV_fqQpXvWBB9jsK3aBdfr8FMRUHPpal-6SqWr36Wree_3j9dNdW-bJ8uflhPA9kDcza3a8Hz-f2J_ASpxsiU</recordid><startdate>201904</startdate><enddate>201904</enddate><creator>Li, Jing</creator><creator>Sun, Jian</creator><creator>Gao, Xinyue</creator><creator>Wu, Zhixin</creator><creator>Shang, Guangdong</creator><general>Springer Berlin Heidelberg</general><general>Springer</general><general>Springer Nature B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>ISR</scope><scope>3V.</scope><scope>7QL</scope><scope>7T7</scope><scope>7WY</scope><scope>7WZ</scope><scope>7X7</scope><scope>7XB</scope><scope>87Z</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8FL</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BEZIV</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FRNLG</scope><scope>FYUFA</scope><scope>F~G</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K60</scope><scope>K6~</scope><scope>K9.</scope><scope>L.-</scope><scope>LK8</scope><scope>M0C</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PQBIZ</scope><scope>PQBZA</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0001-6142-3785</orcidid></search><sort><creationdate>201904</creationdate><title>Coupling ssDNA recombineering with CRISPR-Cas9 for Escherichia coli DnaG mutations</title><author>Li, Jing ; Sun, Jian ; Gao, Xinyue ; Wu, Zhixin ; Shang, Guangdong</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c513t-c33b52dc9ab80dbe320c09baa005e61810ea723895d41b1d6f6c28860824238a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>Adaptive immunity</topic><topic>Antibacterial agents</topic><topic>Bacteria</topic><topic>Biological products</topic><topic>Biomedical and Life Sciences</topic><topic>Biotechnology</topic><topic>Chromosome deletion</topic><topic>Chromosomes</topic><topic>Clonal deletion</topic><topic>Cloning</topic><topic>CRISPR</topic><topic>CRISPR-Cas Systems</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA primase</topic><topic>DNA Primase - genetics</topic><topic>DNA Primase - metabolism</topic><topic>DNA, Bacterial - genetics</topic><topic>DNA, Single-Stranded - genetics</topic><topic>E coli</topic><topic>Editing</topic><topic>Efficiency</topic><topic>Escherichia coli</topic><topic>Escherichia coli - enzymology</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli Proteins - genetics</topic><topic>Escherichia coli Proteins - metabolism</topic><topic>Gene deletion</topic><topic>Gene Editing</topic><topic>Gene mutation</topic><topic>Genes</topic><topic>Genetic aspects</topic><topic>Genetic Engineering - methods</topic><topic>Genetic research</topic><topic>Genome editing</topic><topic>Genome, Bacterial - genetics</topic><topic>Genomes</topic><topic>Genomics</topic><topic>Homologous Recombination</topic><topic>Homology</topic><topic>Immunity</topic><topic>Immunotherapy</topic><topic>Kanamycin</topic><topic>Life Sciences</topic><topic>Metabolic engineering</topic><topic>Methods</topic><topic>Methods and Protocols</topic><topic>Microbial Genetics and Genomics</topic><topic>Microbiology</topic><topic>Mutation</topic><topic>Neomycin</topic><topic>Plasmids - genetics</topic><topic>Plasmids - metabolism</topic><topic>Primase</topic><topic>Structure-function relationships</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Li, Jing</creatorcontrib><creatorcontrib>Sun, Jian</creatorcontrib><creatorcontrib>Gao, Xinyue</creatorcontrib><creatorcontrib>Wu, Zhixin</creatorcontrib><creatorcontrib>Shang, Guangdong</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>ABI/INFORM Collection</collection><collection>ABI/INFORM Global (PDF only)</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>ABI/INFORM Global (Alumni Edition)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ABI/INFORM Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Business Premium Collection</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Business Premium Collection (Alumni)</collection><collection>Health Research Premium Collection</collection><collection>ABI/INFORM Global (Corporate)</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Business Collection (Alumni Edition)</collection><collection>ProQuest Business Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ABI/INFORM Professional Advanced</collection><collection>ProQuest Biological Science Collection</collection><collection>ABI/INFORM Global</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Business</collection><collection>ProQuest One Business (Alumni)</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>MEDLINE - Academic</collection><jtitle>Applied microbiology and biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Li, Jing</au><au>Sun, Jian</au><au>Gao, Xinyue</au><au>Wu, Zhixin</au><au>Shang, Guangdong</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Coupling ssDNA recombineering with CRISPR-Cas9 for Escherichia coli DnaG mutations</atitle><jtitle>Applied microbiology and biotechnology</jtitle><stitle>Appl Microbiol Biotechnol</stitle><addtitle>Appl Microbiol Biotechnol</addtitle><date>2019-04</date><risdate>2019</risdate><volume>103</volume><issue>8</issue><spage>3559</spage><epage>3570</epage><pages>3559-3570</pages><issn>0175-7598</issn><eissn>1432-0614</eissn><abstract>Homologous recombination-based recombineering is a widely used DNA cloning and modification technique; recombineering efficiency improvement would be helpful for high-throughput DNA manipulation.
Escherichia coli
primase DnaG variants, such as DnaG Q576A and DnaG K580A, increase the recombineering efficiency via impairment of the interaction between primase and the replisome and boost the loading of more ssDNA on the replication fork. Bacterial adaptive immunity origin CRISPR-Cas9 is emerging as a powerful genome editing strategy. In this study, ssDNA recombineering and CRISPR-Cas9 were combined for the generation of DnaG variants. The tightly regulated Red operon expression cassette and tightly regulated Cas9 expression cassette were integrated into one chloroamphenicol resistance, p15A replicon-based vector. A self-curing, kanamycin resistance, p15A replicon-based plasmid was applied for the plasmid elimination after genome editing. The genome editing efficiency was as high as 100%. The recombineering efficiency of the strains harboring the DnaG variants was assayed via the kanamycin resistance gene repair as well as the chromosomal gene deletion experiments. The established genome editing strategy will expedite the DnaG structure and function relationship study as well as the metabolic engineering and synthetic biology applications.</abstract><cop>Berlin/Heidelberg</cop><pub>Springer Berlin Heidelberg</pub><pmid>30879090</pmid><doi>10.1007/s00253-019-09744-9</doi><tpages>12</tpages><orcidid>https://orcid.org/0000-0001-6142-3785</orcidid></addata></record> |
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| ispartof | Applied microbiology and biotechnology, 2019-04, Vol.103 (8), p.3559-3570 |
| issn | 0175-7598 1432-0614 |
| language | eng |
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| subjects | Adaptive immunity Antibacterial agents Bacteria Biological products Biomedical and Life Sciences Biotechnology Chromosome deletion Chromosomes Clonal deletion Cloning CRISPR CRISPR-Cas Systems Deoxyribonucleic acid DNA DNA primase DNA Primase - genetics DNA Primase - metabolism DNA, Bacterial - genetics DNA, Single-Stranded - genetics E coli Editing Efficiency Escherichia coli Escherichia coli - enzymology Escherichia coli - genetics Escherichia coli Proteins - genetics Escherichia coli Proteins - metabolism Gene deletion Gene Editing Gene mutation Genes Genetic aspects Genetic Engineering - methods Genetic research Genome editing Genome, Bacterial - genetics Genomes Genomics Homologous Recombination Homology Immunity Immunotherapy Kanamycin Life Sciences Metabolic engineering Methods Methods and Protocols Microbial Genetics and Genomics Microbiology Mutation Neomycin Plasmids - genetics Plasmids - metabolism Primase Structure-function relationships |
| title | Coupling ssDNA recombineering with CRISPR-Cas9 for Escherichia coli DnaG mutations |
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