{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T19:53:56Z","timestamp":1792785236710,"version":"4.5.0"},"reference-count":60,"publisher":"National Academy of Sciences","issue":"4","license":[{"start":{"date-parts":[[2026,1,23]],"date-time":"2026-01-23T00:00:00Z","timestamp":1769126400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"Colton Center for Autoimmunity at Yale","award":["Pilot Award"],"award-info":[{"award-number":["Pilot Award"]}]}],"content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2026,1,27]]},"abstract":"<jats:p>\n                    Bacteriophages have applications in biotechnology, including human and veterinary medicine, agriculture, food safety, and biosecurity. One example of resurging importance is phage therapy, the use of phages to treat antibiotic-resistant bacterial infections. Phage therapy currently requires screening of environmental phages against the infecting strains for each individual patient, a laborious process that limits the development of standardized treatments. To overcome limitations of narrow host range inherent to many native phages, a robust genomic engineering platform is required which permits rapid and dependable genome production and engineering for nonmodel phage. Here, we describe an engineering platform for a phiKMV-like\n                    <jats:italic toggle=\"yes\">Pseudomonas aeruginosa<\/jats:italic>\n                    phage, 41S1 that builds on previous work in the rapid assembly of small genomes through one-pot, High Complexity Golden Gate assembly (HC-GGA). This system divides the 41S1 genome into DNA fragments small enough to be conveniently synthesized and to avoid toxicity during DNA propagation, with all but one maintained in\n                    <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                    . These fragments are readily assembled in a high accuracy, one-pot reaction; phages can be rescued by direct transformation into\n                    <jats:italic toggle=\"yes\">P. aeruginosa<\/jats:italic>\n                    PAO1 or\n                    <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                    10-beta cells. We demonstrate the precise generation of point mutations, DNA insertions, deletions, and the addition of fluorescence reporter genes that are expressed during phage replication. All viable genotypes could be generated with near 100% success rate with minimal screening. This system demonstrates the potential of HC-GGA for the rapid production and engineering of phages and provides a chassis for the development of phages that broadly target the opportunistic human pathogen\n                    <jats:italic toggle=\"yes\">P. aeruginosa<\/jats:italic>\n                    .\n                  <\/jats:p>","DOI":"10.1073\/pnas.2525963123","type":"journal-article","created":{"date-parts":[[2026,1,23]],"date-time":"2026-01-23T18:50:12Z","timestamp":1769194212000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":6,"title":["A fully synthetic Golden Gate assembly system for engineering a\n                    <i>Pseudomonas aeruginosa<\/i>\n                    phiKMV-like phage"],"prefix":"10.1073","volume":"123","author":[{"given":"Andrew P.","family":"Sikkema","sequence":"first","affiliation":[{"name":"Applied Molecular Biology Division, New England Biolabs Research Department","place":["Ipswich"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaitlyn E.","family":"Kortright","sequence":"additional","affiliation":[{"name":"Department of Ecology and Evolutionary Biology, Yale University","place":["New Haven"]},{"name":"Center for Phage Biology & Therapy, Yale University","place":["New Haven"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hemaa","family":"Selvakumar","sequence":"additional","affiliation":[{"name":"Felix Biotechnology","place":["San Francisco"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7402-983X","authenticated-orcid":false,"given":"Jyot","family":"Antani","sequence":"additional","affiliation":[{"name":"Department of Ecology and Evolutionary Biology, Yale University","place":["New Haven"]},{"name":"Center for Phage Biology & Therapy, Yale University","place":["New Haven"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin K.","family":"Chan","sequence":"additional","affiliation":[{"name":"Department of Ecology and Evolutionary Biology, Yale University","place":["New Haven"]},{"name":"Center for Phage Biology & Therapy, Yale University","place":["New Haven"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-2945-6085","authenticated-orcid":false,"given":"Matthew","family":"Davidson","sequence":"additional","affiliation":[{"name":"Department of Ecology and Evolutionary Biology, Yale University","place":["New Haven"]},{"name":"Center for Phage Biology & Therapy, Yale University","place":["New Haven"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Max","family":"Hopkins","sequence":"additional","affiliation":[{"name":"Felix Biotechnology","place":["San Francisco"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin","family":"Newman","sequence":"additional","affiliation":[{"name":"Applied Molecular Biology Division, New England Biolabs Research Department","place":["Ipswich"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7690-3754","authenticated-orcid":false,"given":"Vladimir","family":"Potapov","sequence":"additional","affiliation":[{"name":"Applied Molecular Biology Division, New England Biolabs Research Department","place":["Ipswich"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cecilia A.","family":"Silva-Valenzuela","sequence":"additional","affiliation":[{"name":"Applied Molecular Biology Division, New England Biolabs Research Department","place":["Ipswich"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"S. Kasra","family":"Tabatabaei","sequence":"additional","affiliation":[{"name":"Applied Molecular Biology Division, New England Biolabs Research Department","place":["Ipswich"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert","family":"McBride","sequence":"additional","affiliation":[{"name":"Felix Biotechnology","place":["San Francisco"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3490-7498","authenticated-orcid":false,"given":"Paul E.","family":"Turner","sequence":"additional","affiliation":[{"name":"Department of Ecology and Evolutionary Biology, Yale University","place":["New Haven"]},{"name":"Center for Phage Biology & Therapy, Yale University","place":["New Haven"]},{"name":"Yale School of Medicine, Microbiology Graduate Program","place":["New Haven"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gregory J. S.","family":"Lohman","sequence":"additional","affiliation":[{"name":"Applied Molecular Biology Division, New England Biolabs Research Department","place":["Ipswich"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2026,1,23]]},"reference":[{"key":"e_1_3_4_1_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2017.00981"},{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.3390\/molecules30153132"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.3390\/v15020349"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.4c00681"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2022.11.017"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.00052-20"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkac1037"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.2217\/fmb.13.47"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2019.00954"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibtech.2022.08.008"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0003957"},{"key":"e_1_3_4_12_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gku628"},{"key":"e_1_3_4_13_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-021-86112-6"},{"key":"e_1_3_4_14_2","doi-asserted-by":"publisher","DOI":"10.1093\/synbio\/ysae012"},{"key":"e_1_3_4_15_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-46585-1"},{"key":"e_1_3_4_16_2","doi-asserted-by":"publisher","DOI":"10.3389\/fbioe.2022.869486"},{"key":"e_1_3_4_17_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cels.2015.08.013"},{"key":"e_1_3_4_18_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.94.25.13909"},{"key":"e_1_3_4_19_2","volume-title":"Biotechnology","author":"Thomas C. M.","unstructured":"C. M. Thomas, \u201cGenetic manipulation of bacteria\u201d in Biotechnology Vol. III, H. W. Doelle, S. Rokem, M. Berovic, Eds. (EOLSS Publishers, Oxford, UK, 2009)."},{"key":"e_1_3_4_20_2","doi-asserted-by":"publisher","DOI":"10.1093\/intbio\/zyz005"},{"key":"e_1_3_4_21_2","doi-asserted-by":"publisher","DOI":"10.1128\/mSystems.00659-20"},{"key":"e_1_3_4_22_2","doi-asserted-by":"publisher","DOI":"10.1021\/acssynbio.1c00319"},{"key":"e_1_3_4_23_2","doi-asserted-by":"publisher","DOI":"10.1021\/acssynbio.1c00525"},{"key":"e_1_3_4_24_2","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.03582-24"},{"key":"e_1_3_4_25_2","doi-asserted-by":"publisher","DOI":"10.1002\/cpz1.882"},{"key":"e_1_3_4_26_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0238592"},{"key":"e_1_3_4_27_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2523871122"},{"key":"e_1_3_4_28_2","unstructured":"R. Lavigne Pseudomonas phage phiKMV. National Center for Biotechnology Information. https:\/\/www.ncbi.nlm.nih.gov\/nuccore\/AJ505558.1. Accessed 20 December 2025."},{"key":"e_1_3_4_29_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0042-6822(03)00123-5"},{"key":"e_1_3_4_30_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2020.11.003"},{"key":"e_1_3_4_31_2","doi-asserted-by":"publisher","DOI":"10.2217\/fmb.10.66"},{"key":"e_1_3_4_32_2","first-page":"227","volume-title":"Advances in Virus Research","author":"Krylov V. N.","year":"2014","unstructured":"V. N. Krylov, \u201cBacteriophages of Pseudomonas aeruginosa: Long-Term prospects for use in phage therapy\u201d in Advances in Virus Research, K. Maramorosch, F. A. Murphy, Eds. (Academic Press, 2014), pp. 227\u2013278."},{"key":"e_1_3_4_33_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41392-022-01056-1"},{"key":"e_1_3_4_34_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.biotechadv.2018.11.013"},{"key":"e_1_3_4_35_2","doi-asserted-by":"publisher","DOI":"10.1086\/533452"},{"key":"e_1_3_4_36_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-60327-164-6_2"},{"key":"e_1_3_4_37_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0378-1119(02)00503-6"},{"key":"e_1_3_4_38_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-4220-7_3"},{"key":"e_1_3_4_39_2","doi-asserted-by":"publisher","DOI":"10.1016\/0022-2836(81)90405-8"},{"key":"e_1_3_4_40_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.173.4.1536-1543.1991"},{"key":"e_1_3_4_41_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkx370"},{"key":"e_1_3_4_42_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/10.5.1635"},{"key":"e_1_3_4_43_2","doi-asserted-by":"publisher","DOI":"10.4161\/rna.27766"},{"key":"e_1_3_4_44_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.crmeth.2022.100217"},{"key":"e_1_3_4_45_2","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.00455-21"},{"key":"e_1_3_4_46_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2019.09.015"},{"key":"e_1_3_4_47_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2019.09.062"},{"key":"e_1_3_4_48_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2021.02.006"},{"key":"e_1_3_4_49_2","doi-asserted-by":"publisher","DOI":"10.1099\/mic.0.28431-0"},{"key":"e_1_3_4_50_2","unstructured":"D. P. Pires Pseudomonas phage vB_PaeP_P1G. National Center for Biotechnology Information. https:\/\/www.ncbi.nlm.nih.gov\/nuccore\/OQ230793.1. Accessed 20 December 2025."},{"key":"e_1_3_4_51_2","doi-asserted-by":"publisher","DOI":"10.1186\/1743-422X-8-134"},{"key":"e_1_3_4_52_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-023-06869-w"},{"key":"e_1_3_4_53_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkl600"},{"key":"e_1_3_4_54_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2504853122"},{"key":"e_1_3_4_55_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.173.21.6998-7003.1991"},{"key":"e_1_3_4_56_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.csbj.2022.09.006"},{"key":"e_1_3_4_57_2","unstructured":"A. P. Sikkema Pseudomonas phage 41S1. National Center for Biotechnology Information. https:\/\/www.ncbi.nlm.nih.gov\/nuccore\/PX380000.1. Deposited 17 September 2025."},{"key":"e_1_3_4_58_2","doi-asserted-by":"publisher","DOI":"10.1021\/acssynbio.3c00694"},{"key":"e_1_3_4_59_2","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btaa558"},{"key":"e_1_3_4_60_2","unstructured":"K. Kortright A fully synthetic Golden Gate assembly system for engineering a Pseudomonas aeruginosa phiKMV-like phage [Dataset]. Dryad. https:\/\/doi.org\/10.5061\/dryad.pzgmsbd23. Deposited 4 December 2025."}],"container-title":["Proceedings of the National Academy of Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pnas.org\/doi\/pdf\/10.1073\/pnas.2525963123","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,23]],"date-time":"2026-01-23T18:50:32Z","timestamp":1769194232000},"score":1,"resource":{"primary":{"URL":"https:\/\/pnas.org\/doi\/10.1073\/pnas.2525963123"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,23]]},"references-count":60,"journal-issue":{"issue":"4","published-print":{"date-parts":[[2026,1,27]]}},"alternative-id":["10.1073\/pnas.2525963123"],"URL":"https:\/\/doi.org\/10.1073\/pnas.2525963123","relation":{},"ISSN":["0027-8424","1091-6490"],"issn-type":[{"value":"0027-8424","type":"print"},{"value":"1091-6490","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,23]]},"assertion":[{"value":"2025-09-19","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-12-17","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-01-23","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e2525963123"}}