{"doi":"10.1002/bit.26915","title":"Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in <i>Escherichia coli</i>","abstract":"<jats:title>Abstract</jats:title><jats:p>CRISPR utilizing Cas9 from <jats:italic>Streptococcus pyogenes</jats:italic> (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for \n<jats:italic>Escherichia coli</jats:italic> engineering. To integrate the SpdCas9‐based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single‐guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as \n<jats:italic>Staphylococcus aureus</jats:italic> (SaCas9) and \n<jats:italic>Streptococcus thermophilius CRISPR1</jats:italic> (St1Cas9) as well as Cas12a derived from \n<jats:italic>Francisella novicida</jats:italic> (FnCas12a). At the commonly used \n<jats:italic>E. coli</jats:italic> model genes \n<jats:italic>LacZ</jats:italic>, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10 kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock‐in \n<jats:italic>pyc</jats:italic> and knockout \n<jats:italic>adhE</jats:italic>, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to \n<jats:italic>E. coli</jats:italic> engineering.</jats:p>","journal":"Biotechnology and Bioengineering","year":2019,"id":604123,"datarank":0.5375278407684165,"base_score":3.58351893845611,"endowment":3.58351893845611,"self_citation_contribution":0.5375278407684165,"citation_network_contribution":0.0,"self_endowment_contribution":0.5375278407684165,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":35,"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":1549926,"name":"Meng‐Ying Wu","orcid":null,"position":1,"is_corresponding":false},{"id":1549927,"name":"Mei‐Wei Lin","orcid":null,"position":2,"is_corresponding":false},{"id":1549928,"name":"Mu‐Nung Hsu","orcid":null,"position":3,"is_corresponding":false},{"id":1549929,"name":"Vu Anh Truong","orcid":null,"position":4,"is_corresponding":false},{"id":1549930,"name":"Chih‐Che Shen","orcid":null,"position":5,"is_corresponding":false},{"id":1549931,"name":"Yi Tu","orcid":null,"position":6,"is_corresponding":false},{"id":1549932,"name":"Kuen‐Yuan Hwang","orcid":null,"position":7,"is_corresponding":false},{"id":1549933,"name":"An‐Pang Tu","orcid":null,"position":8,"is_corresponding":false},{"id":1549934,"name":"Yu‐Han Chang","orcid":null,"position":9,"is_corresponding":false},{"id":16363,"name":"Yu‐Chen Hu","orcid":"0000-0002-9997-4467","position":10,"is_corresponding":false},{"id":1549925,"name":"Li‐Yu Sung","orcid":null,"position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in <i>Escherichia coli</i>","abstract":"<jats:title>Abstract</jats:title><jats:p>CRISPR utilizing Cas9 from <jats:italic>Streptococcus pyogenes</jats:italic> (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for \n<jats:italic>Escherichia coli</jats:italic> engineering. To integrate the SpdCas9‐based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single‐guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as \n<jats:italic>Staphylococcus aureus</jats:italic> (SaCas9) and \n<jats:italic>Streptococcus thermophilius CRISPR1</jats:italic> (St1Cas9) as well as Cas12a derived from \n<jats:italic>Francisella novicida</jats:italic> (FnCas12a). At the commonly used \n<jats:italic>E. coli</jats:italic> model genes \n<jats:italic>LacZ</jats:italic>, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10 kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock‐in \n<jats:italic>pyc</jats:italic> and knockout \n<jats:italic>adhE</jats:italic>, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to \n<jats:italic>E. coli</jats:italic> engineering.</jats:p>","is_dataset_classified":null,"base_score":3.58351893845611,"endowment":3.58351893845611,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"30636321","pmcid":null,"openalex_id":"https://openalex.org/W2908865703","authors":[],"funders":[{"funder_name":"Ministry of Science and Technology","grant_id":"106‐2622‐8‐007‐017","title":null},{"funder_name":"Ministry of Science and Technology","grant_id":"106‐2622‐E‐007‐014‐CC1","title":null},{"funder_name":"Ministry of Science and Technology","grant_id":"107‐2622‐E‐007‐003‐CC1","title":null},{"funder_name":"Ministry of Science and Technology","grant_id":"106‐2221‐E‐007‐085‐MY","title":null},{"funder_name":"Chang Gung Memorial Hospital, Linkou","grant_id":"CMRPG3B1542","title":null},{"funder_name":"Chang Gung Memorial Hospital, Linkou","grant_id":"CRRPG3E0061","title":null},{"funder_name":"Chang Gung Memorial Hospital, Linkou","grant_id":"CRRPG3E0172","title":null}],"total_grants":7,"fwci":1.5937,"citation_percentile":0.83319739,"influential_citations":0,"citation_trend":[{"year":2019,"count":6},{"year":2020,"count":5},{"year":2021,"count":5},{"year":2022,"count":5},{"year":2023,"count":4},{"year":2024,"count":6},{"year":2025,"count":2},{"year":2026,"count":2}],"oa_status":"closed","license":"http://onlinelibrary.wiley.com/termsAndConditions#vor","oa_locations":[{"url":"https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1002%2Fbit.26915","host_type":"publisher"},{"url":"https://onlinelibrary.wiley.com/doi/pdf/10.1002/bit.26915","host_type":"publisher"},{"url":"https://onlinelibrary.wiley.com/doi/full-xml/10.1002/bit.26915","host_type":"publisher"},{"url":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/pdf/10.1002/bit.26915","host_type":"publisher"},{"url":"https://doi.org/10.1002/bit.26915","host_type":"journal"},{"url":"https://pubmed.ncbi.nlm.nih.gov/30636321","host_type":"repository"}],"fields_of_study":["CRISPR and Genetic Engineering","RNA and protein synthesis mechanisms","Bacterial Genetics and Biotechnology"],"mesh_terms":["Escherichia coli","Francisella","Genetic Engineering","Staphylococcus aureus","Streptococcus pyogenes","Genome, Bacterial","Gene Knockout Techniques","CRISPR-Cas Systems"],"keywords":["Cas9","CRISPR","CRISPR interference","Genome engineering","Escherichia coli","Nuclease","Guide RNA","Metabolic engineering","Biology","DNA","Computational biology","Gene","Genetics","Crispri","Cas9 Ortholog","St1cas9","Sacas9"],"sdg_mappings":[],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-07-29T23:24:24.715369Z","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":[]}