{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T15:04:38Z","timestamp":1790435078346,"version":"4.1.0"},"reference-count":59,"publisher":"National Academy of Sciences","issue":"46","license":[{"start":{"date-parts":[[2025,11,10]],"date-time":"2025-11-10T00:00:00Z","timestamp":1762732800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"HHS | NIH","doi-asserted-by":"publisher","award":["GM116884"],"award-info":[{"award-number":["GM116884"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Howard Hughes Medica","award":["GT12053"],"award-info":[{"award-number":["GT12053"]}]}],"content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2025,11,18]]},"abstract":"<jats:p>\n                    Bacteriophages show therapeutic promise for treating bacterial pathogens including nontuberculous mycobacteria (NTM). A major impediment is the paucity of therapeutically useful phages and the great variation in the phage infection profiles, especially among\n                    <jats:italic toggle=\"yes\">\n                      <jats:italic toggle=\"yes\">Mycobacterium<\/jats:italic>\n                      abscessus\n                    <\/jats:italic>\n                    clinical isolates. These limitations\u2014together with the abundance of mycobacteriophage genes of unknown function\u2014could be addressed by synthetic genetic construction of viruses in which undesirable genes can be eliminated and genetic payloads can be readily added. However, the relatively high G+C% content of mycobacteriophage genomes (64.1%) can be challenging for DNA synthesis using phosphoramidite chemistry, and the genomes are relatively large (40 to 150 kbp) for assembly and rebooting in a bacterial host. Here, we demonstrate efficient de novo synthesis of high G+C% DNA fragments using terminal deoxynucleotidyl transferase chemistry, the reconstruction of complete mycobacteriophage genomes using High-Complexity Golden Gate Assembly, and efficient rebooting via electroporation into\n                    <jats:italic toggle=\"yes\">\n                      <jats:italic toggle=\"yes\">Mycobacterium<\/jats:italic>\n                      smegmatis\n                    <\/jats:italic>\n                    . Using this approach, we synthesized the genomes of phages BPs (41.9 kbp, 66.6% G+C%) and Bxb1 (50.5 kbp, 63.6% G+C%), and constructed variants carrying targeted mutations or added payloads. Synthetic construction of mycobacteriophages and their derivatives expands the phage repertoire for therapeutic development and provides versatile tools for advancing mycobacterial genetics and phage-based clinical applications.\n                  <\/jats:p>","DOI":"10.1073\/pnas.2523871122","type":"journal-article","created":{"date-parts":[[2025,11,10]],"date-time":"2025-11-10T20:03:22Z","timestamp":1762805002000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":11,"title":["Genome synthesis, assembly, and rebooting of therapeutically useful high G+C% mycobacteriophages"],"prefix":"10.1073","volume":"122","author":[{"given":"Ching-Chung","family":"Ko","sequence":"first","affiliation":[{"name":"Department of Biological Sciences, University of Pittsburgh","place":["Pittsburgh"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew P.","family":"Sikkema","sequence":"additional","affiliation":[{"name":"Research Department, Applied Molecular Biology, New England Biolabs","place":["Ipswich"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael J.","family":"Lauer","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, University of Pittsburgh","place":["Pittsburgh"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizabeth D.","family":"Amarh","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, University of Pittsburgh","place":["Pittsburgh"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rebecca A.","family":"Garlena","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, University of Pittsburgh","place":["Pittsburgh"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel A.","family":"Russell","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, University of Pittsburgh","place":["Pittsburgh"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicole","family":"Chew","sequence":"additional","affiliation":[{"name":"Ansa Biotechnologies","place":["Emeryville"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tan","family":"Li","sequence":"additional","affiliation":[{"name":"Ansa Biotechnologies","place":["Emeryville"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isa Madrigal","family":"Harrison","sequence":"additional","affiliation":[{"name":"Ansa Biotechnologies","place":["Emeryville"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jared","family":"Ellefson","sequence":"additional","affiliation":[{"name":"Ansa Biotechnologies","place":["Emeryville"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6705-6821","authenticated-orcid":false,"given":"Graham F.","family":"Hatfull","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, University of Pittsburgh","place":["Pittsburgh"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gregory J. 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