{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,23]],"date-time":"2025-10-23T17:02:37Z","timestamp":1761238957048,"version":"build-2065373602"},"reference-count":30,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2022,2,11]],"date-time":"2022-02-11T00:00:00Z","timestamp":1644537600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["19K06055, 19K21150, 21K06233"],"award-info":[{"award-number":["19K06055, 19K21150, 21K06233"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100005365","name":"Ehime University","doi-asserted-by":"publisher","award":["Research Unit for Citromics"],"award-info":[{"award-number":["Research Unit for Citromics"]}],"id":[{"id":"10.13039\/501100005365","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>To explore a possible recessive selective marker for future DNA-free genome editing by direct delivery of a CRISPR\/Cas9-single guide RNA (sgRNA) ribonucleoprotein complex, we knocked out homologs of the ArabidopsisMulti-Antibiotic Resistance 1 (MAR1)\/RTS3 gene, mutations of which confer aminoglycoside resistance, in tobacco plants by an efficient Agrobacterium-mediated gene transfer. A Cas9 gene was introduced into Nicotiana tabacum and Nicotiana sylvestris together with an sgRNA gene for one of three different target sequences designed to perfectly match sequences in both S- and T-genome copies of N. tabacumMAR1 homologs (NtMAR1hs). All three sgRNAs directed the introduction of InDels into NtMAR1hs, as demonstrated by CAPS and amplicon sequencing analyses, albeit with varying efficiency. Leaves of regenerated transformant shoots were evaluated for aminoglycoside resistance on shoot-induction media containing different aminoglycoside antibiotics. All transformants tested were as sensitive to those antibiotics as non-transformed control plants, regardless of the mutation rates in NtMAR1hs. The NtMAR1hs\u2013knockout seedlings of the T1 generation showed limited aminoglycoside resistance but failed to form shoots when cultured on shoot-induction media containing kanamycin. The results suggest that, like Arabidopsis MAR1, NtMAR1hs have a role in plants\u2019 sensitivity to aminoglycoside antibiotics, and that tobacco has some additional functional homologs.<\/jats:p>","DOI":"10.3390\/ijms23042006","type":"journal-article","created":{"date-parts":[[2022,2,13]],"date-time":"2022-02-13T21:08:43Z","timestamp":1644786523000},"page":"2006","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Knockout of Tobacco Homologs of Arabidopsis Multi-Antibiotic Resistance 1 Gene Confers a Limited Resistance to Aminoglycoside Antibiotics"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5400-7086","authenticated-orcid":false,"given":"Hafizur","family":"Rahman","sequence":"first","affiliation":[{"name":"The United Graduate School of Agricultural Sciences, Ehime University, Tarumi, Matsuyama 790-8566, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chika","family":"Fukushima","sequence":"additional","affiliation":[{"name":"Faculty of Agriculture, Ehime University, Matsuyama 790-8566, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hidetaka","family":"Kaya","sequence":"additional","affiliation":[{"name":"The United Graduate School of Agricultural Sciences, Ehime University, Tarumi, Matsuyama 790-8566, Japan"},{"name":"Faculty of Agriculture, Ehime University, Matsuyama 790-8566, Japan"},{"name":"Research Unit for Citromics, Ehime University, Matsuyama 790-8566, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Takashi","family":"Yaeno","sequence":"additional","affiliation":[{"name":"The United Graduate School of Agricultural Sciences, Ehime University, Tarumi, Matsuyama 790-8566, Japan"},{"name":"Faculty of Agriculture, Ehime University, Matsuyama 790-8566, Japan"},{"name":"Research Unit for Citromics, Ehime University, Matsuyama 790-8566, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1156-5528","authenticated-orcid":false,"given":"Kappei","family":"Kobayashi","sequence":"additional","affiliation":[{"name":"The United Graduate School of Agricultural Sciences, Ehime University, Tarumi, Matsuyama 790-8566, Japan"},{"name":"Faculty of Agriculture, Ehime University, Matsuyama 790-8566, Japan"},{"name":"Research Unit for Citromics, Ehime University, Matsuyama 790-8566, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,2,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"258","DOI":"10.3389\/fpls.2021.630396","article-title":"Global Regulation of Genetically Modified Crops Amid the Gene Edited Crop Boom\u2014A Review","volume":"12","author":"Turnbull","year":"2021","journal-title":"Front. Plant Sci."},{"key":"ref_2","first-page":"728328","article-title":"Improving Crops for a Changing World","volume":"1","author":"Zhao","year":"2021","journal-title":"Front. Plant Sci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.biotechadv.2014.12.006","article-title":"The CRISPR\/Cas9 system for plant genome editing and beyond","volume":"33","author":"Bortesi","year":"2015","journal-title":"Biotechnol. Adv."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"348","DOI":"10.1111\/tpj.12554","article-title":"Both CRISPR\/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana","volume":"79","author":"Fauser","year":"2014","journal-title":"Plant J."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1621","DOI":"10.1016\/j.cell.2021.01.005","article-title":"Leading Edge Genome engineering for crop improvement and future agriculture","volume":"184","author":"Gao","year":"2021","journal-title":"Cell"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"985","DOI":"10.3389\/fpls.2018.00985","article-title":"CRISPR for crop improvement: An update review","volume":"9","author":"Jaganathan","year":"2018","journal-title":"Front. Plant Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"691","DOI":"10.1038\/nbt.2655","article-title":"Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease","volume":"31","author":"Nekrasov","year":"2013","journal-title":"Nat. Biotechnol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"686","DOI":"10.1038\/nbt.2650","article-title":"Targeted genome modification of crop plants using a CRISPR-Cas system","volume":"31","author":"Shan","year":"2013","journal-title":"Nat. Biotechnol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"688","DOI":"10.1038\/nbt.2654","article-title":"Multiplex and homologous recombination\u2013mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9","volume":"31","author":"Li","year":"2013","journal-title":"Nat. Biotechnol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1162","DOI":"10.1038\/nbt.3389","article-title":"DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins","volume":"33","author":"Woo","year":"2015","journal-title":"Nat. Biotechnol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1584\/jpestics.D12-073","article-title":"Creation of herbicide-tolerant crops by gene targeting","volume":"38","author":"Endo","year":"2013","journal-title":"J. Pestic. Sci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1353","DOI":"10.1007\/s00299-018-2299-0","article-title":"Engineering herbicide-resistant watermelon variety through CRISPR\/Cas9-mediated base-editing","volume":"37","author":"Tian","year":"2018","journal-title":"Plant Cell Rep."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"628","DOI":"10.1016\/j.molp.2016.01.001","article-title":"Engineering Herbicide-Resistant Rice Plants through CRISPR\/Cas9-Mediated Homologous Recombination of Acetolactate Synthase","volume":"9","author":"Sun","year":"2016","journal-title":"Mol. Plant"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"449","DOI":"10.1016\/j.cj.2019.10.001","article-title":"Precise base editing of non-allelic acetolactate synthase genes confers sulfonylurea herbicide resistance in maize","volume":"8","author":"Li","year":"2020","journal-title":"Crop J."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1002\/biot.200800156","article-title":"A novel strategy for obtaining kanamycin resistance in Arabidopsis thaliana by silencing an endogenous gene encoding a putative chloroplast transporter","volume":"4","author":"Aufsatz","year":"2009","journal-title":"Biotechnol. J."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"559","DOI":"10.1104\/pp.109.143487","article-title":"Multiple Antibiotic Resistance in Arabidopsis Is Conferred by Mutations in a Chloroplast-Localized Transport Protein","volume":"151","author":"Conte","year":"2009","journal-title":"Plant Physiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"723384","DOI":"10.3389\/fgeed.2021.723384","article-title":"Loss of MAR1 Function is a Marker for Co-Selection of CRISPR-Induced Mutations in Plants","volume":"3","author":"Rinne","year":"2021","journal-title":"Front. Genome Ed."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1111\/tpj.15286","article-title":"Ferroportin 3 is a dual-targeted mitochondrial\/chloroplast iron exporter necessary for iron homeostasis in Arabidopsis","volume":"107","author":"Kim","year":"2021","journal-title":"Plant J."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1016\/S0006-2952(00)00291-4","article-title":"Antibiotic efflux pumps","volume":"60","author":"Balzi","year":"2000","journal-title":"Biochem. Pharmacol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"49","DOI":"10.4161\/psb.5.1.10142","article-title":"The MAR1 transporter is an opportunistic entry point for antibiotics","volume":"5","author":"Conte","year":"2010","journal-title":"Plant Signal. Behav."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"4331","DOI":"10.1128\/jb.174.13.4331-4337.1992","article-title":"Increase of sensitivity to aminoglycoside antibiotics by polyamine-induced protein (oligopeptide-binding protein) in Escherichia coli","volume":"174","author":"Kashiwagi","year":"1992","journal-title":"J. Bacteriol."},{"key":"ref_22","unstructured":"(2020, March 16). Nicotiana Tabacum Genome Data. Available online: https:\/\/solgenomics.net\/organism\/Nicotiana_tabacum\/genome."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1870","DOI":"10.1093\/molbev\/msw054","article-title":"MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets","volume":"33","author":"Kumar","year":"2016","journal-title":"Mol. Biol. Evol."},{"key":"ref_24","unstructured":"(2020, March 16). CCTop\u2014CRISPR\/Cas9 Target Online Predictor. Available online: https:\/\/cctop.cos.uni-heidelberg.de:8043\/index.html."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"26871","DOI":"10.1038\/srep26871","article-title":"Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9","volume":"6","author":"Kaya","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1676","DOI":"10.1093\/pcp\/pcab123","article-title":"A Simple Heat Treatment Increases SpCas9-Mediated Mutation Efficiency in Arabidopsis","volume":"62","author":"Kurokawa","year":"2021","journal-title":"Plant Cell Physiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"8526","DOI":"10.1073\/pnas.87.21.8526","article-title":"Stable transformation of plastids in higher plants","volume":"87","author":"Svab","year":"1990","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1349","DOI":"10.1093\/nar\/19.6.1349","article-title":"A simple and rapid method for the preparation of plant genomic DNA for PCR analysis","volume":"19","author":"Edwards","year":"1991","journal-title":"Nucleic Acids Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1007\/BF02773348","article-title":"A protocol for rapid DNA extraction from Arabidopsis thaliana for PCR analysis","volume":"22","author":"Kasajima","year":"2004","journal-title":"Plant Mol. Biol. Report."},{"key":"ref_30","unstructured":"(2021, March 02). CRISPResso 2. Available online: https:\/\/crispresso.pinellolab.partners.org\/submission."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/4\/2006\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:17:33Z","timestamp":1760134653000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/4\/2006"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,2,11]]},"references-count":30,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2022,2]]}},"alternative-id":["ijms23042006"],"URL":"https:\/\/doi.org\/10.3390\/ijms23042006","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2022,2,11]]}}}