{"doi":"10.1002/bit.26056","title":"Enhanced integration of large DNA into <i>E. coli</i> chromosome by CRISPR/Cas9","abstract":"<jats:title>ABSTRACT</jats:title><jats:sec><jats:label/><jats:p>Metabolic engineering often necessitates chromosomal integration of multiple genes but integration of large genes into <jats:italic>Escherichia coli</jats:italic> remains difficult. CRISPR/Cas9 is an RNA‐guided system which enables site‐specific induction of double strand break (DSB) and programmable genome editing. Here, we hypothesized that CRISPR/Cas9‐triggered DSB could enhance homologous recombination and augment integration of large DNA into <jats:italic>E. coli</jats:italic> chromosome. We demonstrated that CRISPR/Cas9 system was able to trigger DSB in &gt;98% of cells, leading to subsequent cell death, and identified that mutagenic SOS response played roles in the cell survival. By optimizing experimental conditions and combining the λ‐Red proteins and linear dsDNA, CRISPR/Cas9‐induced DSB enabled homologous recombination of the donor DNA and replacement of <jats:italic>lac</jats:italic>Z gene in the MG1655 strain at efficiencies up to 99%, and allowed high fidelity, scarless integration of 2.4, 3.9, 5.4, and 7.0 kb DNA at efficiencies approaching 91%, 92%, 71%, and 61%, respectively. The CRISPR/Cas9‐assisted gene integration also functioned in different <jats:italic>E. coli</jats:italic> strains including BL21 (DE3) and W albeit at different efficiencies. Taken together, our methodology facilitated precise integration of dsDNA as large as 7 kb into <jats:italic>E. coli</jats:italic> with efficiencies exceeding 60%, thus significantly ameliorating the editing efficiency and overcoming the size limit of integration using the commonly adopted recombineering approach. Biotechnol. Bioeng. 2017;114: 172–183. © 2016 Wiley Periodicals, Inc.</jats:p></jats:sec>","journal":"Biotechnology and Bioengineering","year":2017,"id":656779,"datarank":0.736898232860408,"base_score":4.912654885736052,"endowment":4.912654885736052,"self_citation_contribution":0.736898232860408,"citation_network_contribution":0.0,"self_endowment_contribution":0.736898232860408,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":135,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1714418,"name":"I‐Hsin Yeh","orcid":null,"position":1,"is_corresponding":false},{"id":1549925,"name":"Li‐Yu Sung","orcid":null,"position":2,"is_corresponding":false},{"id":1549926,"name":"Meng‐Ying Wu","orcid":null,"position":3,"is_corresponding":false},{"id":1714419,"name":"Yun‐Peng Chao","orcid":null,"position":4,"is_corresponding":false},{"id":1714420,"name":"I‐Son Ng","orcid":null,"position":5,"is_corresponding":false},{"id":16363,"name":"Yu‐Chen Hu","orcid":"0000-0002-9997-4467","position":6,"is_corresponding":false},{"id":1714417,"name":"Mu‐En Chung","orcid":null,"position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Enhanced integration of large DNA into <i>E. coli</i> chromosome by CRISPR/Cas9","abstract":"<jats:title>ABSTRACT</jats:title><jats:sec><jats:label/><jats:p>Metabolic engineering often necessitates chromosomal integration of multiple genes but integration of large genes into <jats:italic>Escherichia coli</jats:italic> remains difficult. CRISPR/Cas9 is an RNA‐guided system which enables site‐specific induction of double strand break (DSB) and programmable genome editing. Here, we hypothesized that CRISPR/Cas9‐triggered DSB could enhance homologous recombination and augment integration of large DNA into <jats:italic>E. coli</jats:italic> chromosome. We demonstrated that CRISPR/Cas9 system was able to trigger DSB in &gt;98% of cells, leading to subsequent cell death, and identified that mutagenic SOS response played roles in the cell survival. By optimizing experimental conditions and combining the λ‐Red proteins and linear dsDNA, CRISPR/Cas9‐induced DSB enabled homologous recombination of the donor DNA and replacement of <jats:italic>lac</jats:italic>Z gene in the MG1655 strain at efficiencies up to 99%, and allowed high fidelity, scarless integration of 2.4, 3.9, 5.4, and 7.0 kb DNA at efficiencies approaching 91%, 92%, 71%, and 61%, respectively. The CRISPR/Cas9‐assisted gene integration also functioned in different <jats:italic>E. coli</jats:italic> strains including BL21 (DE3) and W albeit at different efficiencies. Taken together, our methodology facilitated precise integration of dsDNA as large as 7 kb into <jats:italic>E. coli</jats:italic> with efficiencies exceeding 60%, thus significantly ameliorating the editing efficiency and overcoming the size limit of integration using the commonly adopted recombineering approach. Biotechnol. Bioeng. 2017;114: 172–183. © 2016 Wiley Periodicals, Inc.</jats:p></jats:sec>","is_dataset_classified":null,"base_score":4.912654885736052,"endowment":4.912654885736052,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"27454445","pmcid":null,"openalex_id":"https://openalex.org/W2498972343","authors":[],"funders":[{"funder_name":"Ministry of Science and Technology, Taiwan","grant_id":"103B7028J2","title":null}],"total_grants":1,"fwci":6.7325,"citation_percentile":0.97384763,"influential_citations":0,"citation_trend":[{"year":2016,"count":3},{"year":2017,"count":13},{"year":2018,"count":10},{"year":2019,"count":16},{"year":2020,"count":17},{"year":2021,"count":26},{"year":2022,"count":10},{"year":2023,"count":8},{"year":2024,"count":26},{"year":2025,"count":3},{"year":2026,"count":3}],"oa_status":"closed","license":"http://onlinelibrary.wiley.com/termsAndConditions#vor","oa_locations":[{"url":"https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1002%2Fbit.26056","host_type":"publisher"},{"url":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/pdf/10.1002/bit.26056","host_type":"publisher"},{"url":"https://doi.org/10.1002/bit.26056","host_type":"journal"},{"url":"https://pubmed.ncbi.nlm.nih.gov/27454445","host_type":"repository"}],"fields_of_study":["CRISPR and Genetic Engineering","Animal Genetics and Reproduction","Bacterial Genetics and Biotechnology","CRISPR-Cas Systems","Cell Survival","DNA","DNA Breaks, Double-Stranded","Escherichia coli","Gene Editing","Metabolic Engineering","Plasmids","SOS Response, Genetics"],"mesh_terms":["Gene Editing","Cell Survival","DNA","Escherichia coli","Plasmids","SOS Response, Genetics","DNA Breaks, Double-Stranded","Metabolic Engineering","CRISPR-Cas Systems"],"keywords":["Recombineering","CRISPR","Cas9","Genome editing","Homologous recombination","Escherichia coli","Biology","Gene","DNA","Genome engineering","CRISPR interference","SOS response","Genetics","Computational biology","E. coli","Integration","Double strand break","metabolic engineering","Crispr/cas9"],"sdg_mappings":[],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-08-11T22:14:33.484570Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}