{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T15:03:03Z","timestamp":1790434983773,"version":"4.1.0"},"reference-count":57,"publisher":"National Academy of Sciences","issue":"30","license":[{"start":{"date-parts":[[2025,7,25]],"date-time":"2025-07-25T00:00:00Z","timestamp":1753401600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000015","name":"U.S. Department of Energy","doi-asserted-by":"publisher","award":["DE-AC02-05CH11231"],"award-info":[{"award-number":["DE-AC02-05CH11231"]}],"id":[{"id":"10.13039\/100000015","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2025,7,29]]},"abstract":"<jats:p>\n                    Phage \u03bb, a well-characterized temperate phage, has been recently leveraged for bacterial genome editing by selectively delivering base editors into targeted bacterial species. We extend this concept by engineering phage \u03bb to deliver CRISPR-guided transposases, accomplishing large insertions and targeted gene disruptions. To achieve this, we engineered phage \u03bb using homologous recombination paired with Cas13a-based counterselection for precise phage modifications. Initially, we established the utility of Cas13a in phage \u03bb by conducting minimal recoding edits, deletions, and insertions. Subsequently, we scaled up the engineering to embed the comprehensive DNA-editing CRISPR-Cas transposase (DART) system within the phage genome, creating \u03bb-DART phages. These modified \u03bb-DART phages were then employed to infect\n                    <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                    , generating CRISPR RNA-guided transposition events in the host genome. Applying our engineered \u03bb-DART phages to monocultures and a mixed bacterial community comprising three genera led to efficient, precise, and specific gene knockouts and insertions in the targeted\n                    <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                    cells, achieving editing efficiencies surpassing 50% of the population. This research enhances phage-mediated genome editing by enabling efficient in situ gene integrations in bacteria, offering an avenue for further application in microbial community contexts. This scalable method enables flexible microbial genome editing in situ to manipulate the function and composition of diverse ecosystems.\n                  <\/jats:p>","DOI":"10.1073\/pnas.2504853122","type":"journal-article","created":{"date-parts":[[2025,7,25]],"date-time":"2025-07-25T16:54:45Z","timestamp":1753462485000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":31,"title":["Phage-based delivery of CRISPR-associated transposases for targeted bacterial editing"],"prefix":"10.1073","volume":"122","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1893-9599","authenticated-orcid":false,"given":"Avery","family":"Roberts","sequence":"first","affiliation":[{"name":"Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University","place":["Raleigh"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7488-3040","authenticated-orcid":false,"given":"Benjamin A.","family":"Adler","sequence":"additional","affiliation":[{"name":"Innovative Genomics Institute, University of California","place":["Berkeley"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2948-2846","authenticated-orcid":false,"given":"Brady F.","family":"Cress","sequence":"additional","affiliation":[{"name":"Innovative Genomics Institute, University of California","place":["Berkeley"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9161-999X","authenticated-orcid":false,"given":"Jennifer A.","family":"Doudna","sequence":"additional","affiliation":[{"name":"Innovative Genomics Institute, University of California","place":["Berkeley"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0648-3504","authenticated-orcid":false,"given":"Rodolphe","family":"Barrangou","sequence":"additional","affiliation":[{"name":"Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University","place":["Raleigh"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2025,7,25]]},"reference":[{"key":"e_1_3_4_1_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41396-019-0484-y"},{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-020-0718-6"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1098\/rstb.2019.0245"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tig.2016.01.005"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jnutbio.2017.08.006"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1080\/19490976.2024.2356279"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-023-00933-y"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.96.5.2192"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.micro.55.1.437"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.fm.2015.06.006"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.3390\/v5030806"},{"key":"e_1_3_4_12_2","doi-asserted-by":"publisher","DOI":"10.1186\/s12951-024-02576-4"},{"key":"e_1_3_4_13_2","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.00019-20"},{"key":"e_1_3_4_14_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-023-01759-y"},{"key":"e_1_3_4_15_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0704624104"},{"key":"e_1_3_4_16_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2206744119"},{"key":"e_1_3_4_17_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-024-07681-w"},{"key":"e_1_3_4_18_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1138140"},{"key":"e_1_3_4_19_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibtech.2022.08.008"},{"key":"e_1_3_4_20_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2020.11.012"},{"key":"e_1_3_4_21_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41576-018-0059-1"},{"key":"e_1_3_4_22_2","doi-asserted-by":"publisher","DOI":"10.1021\/acssynbio.7b00308"},{"key":"e_1_3_4_23_2","doi-asserted-by":"publisher","DOI":"10.4014\/jmb.0811.669"},{"key":"e_1_3_4_24_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-021-01014-7"},{"key":"e_1_3_4_25_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-019-1323-z"},{"key":"e_1_3_4_26_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-020-00745-y"},{"key":"e_1_3_4_27_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2021.12.021"},{"key":"e_1_3_4_28_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkac985"},{"key":"e_1_3_4_29_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2015.02.010"},{"key":"e_1_3_4_30_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-019-1257-5"},{"key":"e_1_3_4_31_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-022-01258-x"},{"key":"e_1_3_4_32_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-022-01243-4"},{"key":"e_1_3_4_33_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.ge.06.120172.001315"},{"key":"e_1_3_4_34_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.155.3.1130-1137.1983"},{"key":"e_1_3_4_35_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.micro.54.1.799"},{"key":"e_1_3_4_36_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0022-2836(66)80269-3"},{"key":"e_1_3_4_37_2","first-page":"113","volume-title":"Prophage Insertion and Excision in The Bacteriophage Lambda","author":"Gottesman M.","year":"1971","unstructured":"M. Gottesman, R. Weisbeg, Prophage Insertion and Excision in The Bacteriophage Lambda (Cold Spring Harbor Laboratory, 1971), pp. 113\u2013138."},{"key":"e_1_3_4_38_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1004217"},{"key":"e_1_3_4_39_2","doi-asserted-by":"publisher","DOI":"10.1128\/mr.56.4.577-591.1992"},{"key":"e_1_3_4_40_2","doi-asserted-by":"publisher","DOI":"10.1016\/0042-6822(77)90425-1"},{"key":"e_1_3_4_41_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.3002416"},{"key":"e_1_3_4_42_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-025-01935-7"},{"key":"e_1_3_4_43_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.plasmid.2010.12.004"},{"key":"e_1_3_4_44_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-022-05059-4"},{"key":"e_1_3_4_45_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkad270"},{"key":"e_1_3_4_46_2","doi-asserted-by":"crossref","unstructured":"L. C. T. Song Identification of proteins influencing CRISPR-associated transposases for enhanced genome editing. biorXiv [Preprint] (2024) https:\/\/doi.org\/10.1101\/2024.09.11.612086 (Accessed 12 November 2024).","DOI":"10.1101\/2024.09.11.612086"},{"key":"e_1_3_4_47_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41396-021-00912-2"},{"key":"e_1_3_4_48_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-024-01832-5"},{"key":"e_1_3_4_49_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkz085"},{"key":"e_1_3_4_50_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-8940-9"},{"key":"e_1_3_4_51_2","doi-asserted-by":"publisher","DOI":"10.1201\/9780203491751"},{"key":"e_1_3_4_52_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0016765"},{"key":"e_1_3_4_53_2","doi-asserted-by":"publisher","DOI":"10.1093\/nargab\/lqaf009"},{"key":"e_1_3_4_54_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-019-0072-8"},{"key":"e_1_3_4_55_2","doi-asserted-by":"publisher","DOI":"10.1099\/mgen.0.000685"},{"key":"e_1_3_4_56_2","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/bty191"},{"key":"e_1_3_4_57_2","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btaa213"}],"container-title":["Proceedings of the National Academy of Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pnas.org\/doi\/pdf\/10.1073\/pnas.2504853122","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pnas.org\/doi\/pdf\/10.1073\/pnas.2504853122","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,7,25]],"date-time":"2025-07-25T16:54:50Z","timestamp":1753462490000},"score":1,"resource":{"primary":{"URL":"https:\/\/pnas.org\/doi\/10.1073\/pnas.2504853122"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,25]]},"references-count":57,"journal-issue":{"issue":"30","published-print":{"date-parts":[[2025,7,29]]}},"alternative-id":["10.1073\/pnas.2504853122"],"URL":"https:\/\/doi.org\/10.1073\/pnas.2504853122","relation":{},"ISSN":["0027-8424","1091-6490"],"issn-type":[{"value":"0027-8424","type":"print"},{"value":"1091-6490","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,7,25]]},"assertion":[{"value":"2025-03-02","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-06-04","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-07-25","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e2504853122"}}