{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T05:07:13Z","timestamp":1779340033328,"version":"3.51.4"},"reference-count":46,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2022,11,16]],"date-time":"2022-11-16T00:00:00Z","timestamp":1668556800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Witten\/Herdecke University","award":["IFF2017-17"],"award-info":[{"award-number":["IFF2017-17"]}]},{"name":"Witten\/Herdecke University","award":["EH 192\/5-1"],"award-info":[{"award-number":["EH 192\/5-1"]}]},{"DOI":"10.13039\/501100001659","name":"DFG","doi-asserted-by":"publisher","award":["IFF2017-17"],"award-info":[{"award-number":["IFF2017-17"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001659","name":"DFG","doi-asserted-by":"publisher","award":["EH 192\/5-1"],"award-info":[{"award-number":["EH 192\/5-1"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>Adenoviruses are non-enveloped linear double-stranded DNA viruses with over 100 types in humans. Adenovirus vectors have gained tremendous attention as gene delivery vehicles, as vaccine vectors and as oncolytic viruses. Although various methods have been used to generate adenoviral vectors, the vector-producing process remains technically challenging regarding efficacious genome modification. Based on our previously reported adenoviral genome modification streamline via linear\u2013circular homologous recombination, we further develop an HEHR (combining Homing Endonucleases and Homologous Recombination) method to engineer adenoviral genomes more efficiently. I-PpoI, a rare endonuclease encoded by a group I intron, was introduced into the previously described ccdB counter-selection marker. We found that the I-PpoI pre-treatment of counter-selection containing parental plasmid increased the homologous recombination efficiency up to 100%. The flanking of the counter-selection marker with either single or double I-PpoI sites showed enhanced efficacy. In addition, we constructed a third counter-selection marker flanked by an alternative restriction enzyme: AbsI, which could be applied in case the I-PpoI site already existed in the transgene cassette that was previously inserted in the adenovirus genome. Together, HEHR can be applied for seamless sequence replacements, deletions and insertions. The advantages of HEHR in seamless mutagenesis will facilitate rational design of adenoviral vectors for diverse purposes.<\/jats:p>","DOI":"10.3390\/genes13112129","type":"journal-article","created":{"date-parts":[[2022,11,17]],"date-time":"2022-11-17T03:24:34Z","timestamp":1668655474000},"page":"2129","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["HEHR: Homing Endonuclease-Mediated Homologous Recombination for Efficient Adenovirus Genome Engineering"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2242-2450","authenticated-orcid":false,"given":"Katrin","family":"Schr\u00f6er","sequence":"first","affiliation":[{"name":"Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), Department of Human Medicine, Faculty of Health, Witten\/Herdecke University, 58448 Witten, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fatima","family":"Arakrak","sequence":"additional","affiliation":[{"name":"Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), Department of Human Medicine, Faculty of Health, Witten\/Herdecke University, 58448 Witten, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Annika","family":"Bremke","sequence":"additional","affiliation":[{"name":"Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), Department of Human Medicine, Faculty of Health, Witten\/Herdecke University, 58448 Witten, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anja","family":"Ehrhardt","sequence":"additional","affiliation":[{"name":"Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), Department of Human Medicine, Faculty of Health, Witten\/Herdecke University, 58448 Witten, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6243-6286","authenticated-orcid":false,"given":"Wenli","family":"Zhang","sequence":"additional","affiliation":[{"name":"Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), Department of Human Medicine, Faculty of Health, Witten\/Herdecke University, 58448 Witten, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,11,16]]},"reference":[{"key":"ref_1","unstructured":"Journal of Gene Medicine (2022, November 09). Vectors Used for Gene Transfer in Gene Therapy Clinical Trials. Available online: https:\/\/a873679.fmphost.com\/fmi\/webd\/GTCT."},{"key":"ref_2","unstructured":"Human Adenovirus Working Group (2022, November 09). Human Adenovirus Types. Available online: http:\/\/hadvwg.gmu.edu\/."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"675","DOI":"10.1007\/s11262-017-1487-2","article-title":"Getting genetic access to natural adenovirus genomes to explore vector diversity","volume":"53","author":"Zhang","year":"2017","journal-title":"Virus Genes"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Zhang, W., Mese, K., Schellhorn, S., Bahlmann, N., Mach, N., Bunz, O., Dhingra, A., Hage, E., Lafon, M.E., and Wodrich, H. (2020). High-Throughput Cloning and Characterization of Emerging Adenovirus Types 70, 73, 74, and 75. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21176370"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"4654","DOI":"10.1128\/JVI.02696-06","article-title":"Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D","volume":"81","author":"Abbink","year":"2007","journal-title":"J. Virol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1698","DOI":"10.1016\/j.celrep.2017.05.008","article-title":"An Engineered Virus Library as a Resource for the Spectrum-wide Exploration of Virus and Vector Diversity","volume":"19","author":"Zhang","year":"2017","journal-title":"Cell Rep."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1089\/hgtb.2018.044","article-title":"Novel Vector Construction Based on Alternative Adenovirus Types via Homologous Recombination","volume":"29","author":"Zhang","year":"2018","journal-title":"Hum. Gene Ther. Methods"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Liu, J., Boehme, P., Zhang, W., Fu, J., Yumul, R., Mese, K., Tsoukas, R., Solanki, M., Kaufmann, M., and Lu, R. (2018). Human adenovirus type 17 from species D transduces endothelial cells and human CD46 is involved in cell entry. Sci. Rep., 8.","DOI":"10.1038\/s41598-018-31713-x"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1016\/j.omtm.2019.07.001","article-title":"Enhanced Tropism of Species B1 Adenoviral-Based Vectors for Primary Human Airway Epithelial Cells","volume":"14","author":"Li","year":"2019","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Jung, J.M., Ching, W., Baumdick, M.E., Hofmann-Sieber, H., Bosse, J.B., Koyro, T., Moller, K.J., Wegner, L., Niehrs, A., and Russu, K. (2021). KIR3DS1 directs NK cell-mediated protection against human adenovirus infections. Sci. Immunol., 6.","DOI":"10.1126\/sciimmunol.abe2942"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"460","DOI":"10.1158\/1535-7163.MCT-21-0240","article-title":"Novel Group C Oncolytic Adenoviruses Carrying a miRNA Inhibitor Demonstrate Enhanced Oncolytic Activity In Vitro and In Vivo","volume":"21","author":"Doerner","year":"2022","journal-title":"Mol. Cancer Ther."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Tsoukas, R.L., Volkwein, W., Gao, J., Schiwon, M., Bahlmann, N., Dittmar, T., Hagedorn, C., Ehrke-Schulz, E., Zhang, W., and Baiker, A. (2022). A Human In Vitro Model to Study Adenoviral Receptors and Virus Cell Interactions. Cells, 11.","DOI":"10.3390\/cells11050841"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1016\/0092-8674(83)90502-0","article-title":"The origin of replication of plasmid p15A and comparative studies on the nucleotide sequences around the origin of related plasmids","volume":"32","author":"Selzer","year":"1983","journal-title":"Cell"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"816","DOI":"10.1126\/science.1225829","article-title":"A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity","volume":"337","author":"Jinek","year":"2012","journal-title":"Science"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"5809","DOI":"10.1128\/MCB.18.10.5809","article-title":"I-PpoI, the endonuclease encoded by the group I intron PpLSU3, is expressed from an RNA polymerase I transcript","volume":"18","author":"Lin","year":"1998","journal-title":"Mol. Cell Biol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Wang, H., Bian, X., Xia, L., Ding, X., Muller, R., Zhang, Y., Fu, J., and Stewart, A.F. (2014). Improved seamless mutagenesis by recombineering using ccdB for counterselection. Nucleic Acids Res., 42.","DOI":"10.1093\/nar\/gkt1339"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1016\/j.plasmid.2013.02.003","article-title":"Plasmid R6K replication control","volume":"69","author":"Rakowski","year":"2013","journal-title":"Plasmid"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2574","DOI":"10.1073\/pnas.82.9.2574","article-title":"Replication initiator protein of plasmid R6K autoregulates its own synthesis at the transcriptional step","volume":"82","author":"Kelley","year":"1985","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"e150","DOI":"10.1093\/nar\/gks643","article-title":"Transposon-mediated BAC transgenesis in human ES cells","volume":"40","author":"Rostovskaya","year":"2012","journal-title":"Nucleic Acids Res"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s15010-020-01484-7","article-title":"Adenovirus diseases: A systematic review and meta-analysis of 228 case reports","volume":"49","author":"Gu","year":"2020","journal-title":"Infection"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1080\/14760584.2019.1588113","article-title":"A review of 65 years of human adenovirus seroprevalence","volume":"18","author":"Mennechet","year":"2019","journal-title":"Expert Rev. Vaccines"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"3571","DOI":"10.1002\/1873-3468.13576","article-title":"Adenovirus persistence, reactivation, and clinical management","volume":"593","author":"Lion","year":"2019","journal-title":"FEBS Lett."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1556\/EuJMI.4.2014.1.2","article-title":"Human adenovirus: Viral pathogen with increasing importance","volume":"4","author":"Ghebremedhin","year":"2014","journal-title":"Eur. J. Microbiol. Immunol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"3395","DOI":"10.1002\/1873-3468.13700","article-title":"Lessons learned from adenovirus (1970\u20132019)","volume":"593","author":"Nemerow","year":"2019","journal-title":"FEBS Lett."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Sakurai, F., Tachibana, M., and Mizuguchi, H. (2022). Adenovirus vector-based vaccine for infectious diseases. Drug Metab. Pharmacokinet., 42.","DOI":"10.1016\/j.dmpk.2021.100432"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1351","DOI":"10.1016\/S0140-6736(21)00628-0","article-title":"Efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine against SARS-CoV-2 variant of concern 202012\/01 (B.1.1.7): An exploratory analysis of a randomised controlled trial","volume":"397","author":"Emary","year":"2021","journal-title":"Lancet"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1016\/S0140-6736(21)00234-8","article-title":"Safety and efficacy of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine: An interim analysis of a randomised controlled phase 3 trial in Russia","volume":"397","author":"Logunov","year":"2021","journal-title":"Lancet"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"479","DOI":"10.1016\/S0140-6736(20)31605-6","article-title":"Immunogenicity and safety of a recombinant adenovirus type-5-vectored COVID-19 vaccine in healthy adults aged 18 years or older: A randomised, double-blind, placebo-controlled, phase 2 trial","volume":"396","author":"Zhu","year":"2020","journal-title":"Lancet"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"13207","DOI":"10.1128\/JVI.78.23.13207-13215.2004","article-title":"Novel replication-incompetent vector derived from adenovirus type 11 (Ad11) for vaccination and gene therapy: Low seroprevalence and non-cross-reactivity with Ad5","volume":"78","author":"Holterman","year":"2004","journal-title":"J. Virol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"8263","DOI":"10.1128\/JVI.77.15.8263-8271.2003","article-title":"Replication-deficient human adenovirus type 35 vectors for gene transfer and vaccination: Efficient human cell infection and bypass of preexisting adenovirus immunity","volume":"77","author":"Vogels","year":"2003","journal-title":"J. Virol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"384","DOI":"10.1016\/S0042-6822(03)00161-2","article-title":"Development of adenovirus serotype 35 as a gene transfer vector","volume":"311","author":"Ganesh","year":"2003","journal-title":"Virology"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"8946","DOI":"10.1128\/jvi.71.11.8946-8951.1997","article-title":"Construction of an adenovirus type 7a E1A- vector","volume":"71","author":"Abrahamsen","year":"1997","journal-title":"J. Virol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"6186","DOI":"10.1073\/pnas.91.13.6186","article-title":"Efficient manipulation of the human adenovirus genome as an infectious yeast artificial chromosome clone","volume":"91","author":"Ketner","year":"1994","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/j.jviromet.2018.04.001","article-title":"DNA assembly technique simplifies the construction of infectious clone of fowl adenovirus","volume":"257","author":"Zou","year":"2018","journal-title":"J. Virol. Methods"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Pan, H., Yan, Y., Zhang, J., Zhao, S., Feng, L., Ou, J., Cao, N., Li, M., Zhao, W., and Wan, C. (2018). Rapid Construction of a Replication-Competent Infectious Clone of Human Adenovirus Type 14 by Gibson Assembly. Viruses, 10.","DOI":"10.3390\/v10100568"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"e89","DOI":"10.1093\/nar\/gku289","article-title":"Efficient chromosomal gene modification with CRISPR\/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells","volume":"42","author":"Bottcher","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1038\/2417","article-title":"A new logic for DNA engineering using recombination in Escherichia coli","volume":"20","author":"Zhang","year":"1998","journal-title":"Nat. Genet."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"440","DOI":"10.1038\/nbt.2183","article-title":"Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting","volume":"30","author":"Fu","year":"2012","journal-title":"Nat. Biotechnol."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Zhang, Y., Muyrers, J.P., Rientjes, J., and Stewart, A.F. (2003). Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells. BMC Mol. Biol., 4.","DOI":"10.1186\/1471-2199-4-1"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1111\/j.1365-2958.2008.06133.x","article-title":"Involvement of DNA replication in phage lambda Red-mediated homologous recombination","volume":"68","author":"Poteete","year":"2008","journal-title":"Mol. Microbiol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1038\/nmeth.1318","article-title":"Enzymatic assembly of DNA molecules up to several hundred kilobases","volume":"6","author":"Gibson","year":"2009","journal-title":"Nat. Methods"},{"key":"ref_42","unstructured":"(2022, November 09). NEBUILDER HIFI DNA ASSEMBLY\u00ae Removal of 3\u2019 End Mismatches. Available online: https:\/\/international.neb.com\/tools-and-resources\/video-library\/nebuilder-hifi-dna-assembly-removal-of-3-end-mismatches."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Guo, X., Sun, Y., Chen, J., Zou, X., Hou, W., Tan, W., Hung, T., and Lu, Z. (2022). Restriction-Assembly: A Solution to Construct Novel Adenovirus Vector. Viruses, 14.","DOI":"10.3390\/v14030546"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1016\/j.jbiotec.2019.11.009","article-title":"Site-directed modification of adenoviral vector with combined DNA assembly and restriction-ligation cloning","volume":"307","author":"Guo","year":"2020","journal-title":"J. Biotechnol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"6127","DOI":"10.1093\/nar\/16.13.6127","article-title":"High efficiency transformation of E. coli by high voltage electroporation","volume":"16","author":"Dower","year":"1988","journal-title":"Nucleic Acids Res."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1023\/A:1022575601492","article-title":"Properties of L-arabinose isomerase from Escherichia coli as biocatalyst for tagatose production","volume":"19","author":"Yoon","year":"2003","journal-title":"World J. Microbiol. Biotechnol."}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/13\/11\/2129\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:19:27Z","timestamp":1760145567000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/13\/11\/2129"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,11,16]]},"references-count":46,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2022,11]]}},"alternative-id":["genes13112129"],"URL":"https:\/\/doi.org\/10.3390\/genes13112129","relation":{},"ISSN":["2073-4425"],"issn-type":[{"value":"2073-4425","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,11,16]]}}}