{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T10:15:14Z","timestamp":1788516914638,"version":"build-2803163510"},"reference-count":26,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T00:00:00Z","timestamp":1788480000000},"content-version":"vor","delay-in-days":3,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/100005156","name":"Alexander von Humboldt-Stiftung","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100005156","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Physiologia Plantarum"],"published-print":{"date-parts":[[2026,9]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    Functional characterization of a large number of rice genes remains a major challenge despite the availability of genome sequences and large\u2010scale transcriptomic datasets. CRISPR\u2010Cas9 library is a powerful approach for high\u2010throughput targeted mutagenesis; however, its application in indica rice cultivars remains limited due to low transformation and regeneration efficiencies. In this study, we developed a CRISPR\u2010Cas9 library targeting 12,000 rice genes and evaluated its utility for functional genomics in the indica cultivar MTU\u20101010. Sanger sequencing and NGS analysis of the plasmid library revealed high sgRNA coverage and more than 80% accuracy. Transformation of the developed library into the indica cultivar MTU\u20101010 resulted in a high target editing efficiency, with 90% of analyzed transgenic plants carrying mutations at the intended target site. Functional analysis of one homozygous mutant identified a previously uncharacterized role for\n                    <jats:italic>OsOPR5<\/jats:italic>\n                    (\n                    <jats:italic>LOC_Os06g11210<\/jats:italic>\n                    ), a member of the 12\u2010oxophytodienoate reductase family in root architecture. The\n                    <jats:italic>opr5<\/jats:italic>\n                    mutants exhibited significant reductions in lateral root number, seminal and crown root number, and root length, demonstrating that\n                    <jats:italic>OsOPR5<\/jats:italic>\n                    positively regulates root system architecture in rice. Notably, endogenous jasmonic acid (JA) and JA\u2010isoleucine levels were not significantly altered in the mutant, suggesting potential functional specialization or redundancy among rice OPR family members for JA accumulation. The root system architecture is a key determinant of water and nutrient acquisition; our results suggest that\n                    <jats:italic>OsOPR5<\/jats:italic>\n                    may play an important role in adaptation under adverse environmental conditions. Collectively, this study establishes an efficient genome\u2010editing platform for indica rice and identifies\n                    <jats:italic>OsOPR5<\/jats:italic>\n                    as a novel regulator of root development.\n                  <\/jats:p>","DOI":"10.1111\/ppl.71097","type":"journal-article","created":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T09:41:56Z","timestamp":1788514916000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Establishment of a\n                    <scp>CRISPR<\/scp>\n                    \u2010Cas9 Library for Indica Rice and Identification of\n                    <scp>\n                      <i>OsOPR5<\/i>\n                    <\/scp>\n                    (\n                    <i>LOC_Os06g11210<\/i>\n                    ) as a Regulator of Root Architecture"],"prefix":"10.1111","volume":"178","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-4066-4506","authenticated-orcid":false,"given":"Sankhadeep","family":"Chowdhury","sequence":"first","affiliation":[{"name":"BRIC\u2010National Institute of Plant Genome Research (NIPGR)  New Delhi India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-8872-6979","authenticated-orcid":false,"given":"Suprava Priyadarsini","family":"Nayak","sequence":"additional","affiliation":[{"name":"BRIC\u2010National Institute of Plant Genome Research (NIPGR)  New Delhi India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-8727-4023","authenticated-orcid":false,"given":"Rajashree","family":"Pattanayak","sequence":"additional","affiliation":[{"name":"BRIC\u2010National Institute of Plant Genome Research (NIPGR)  New Delhi India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shaswati","family":"Sardar","sequence":"additional","affiliation":[{"name":"BRIC\u2010National Institute of Plant Genome Research (NIPGR)  New Delhi India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-5317-7268","authenticated-orcid":false,"given":"Ashis","family":"Majhi","sequence":"additional","affiliation":[{"name":"BRIC\u2010National Institute of Plant Genome Research (NIPGR)  New Delhi India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-0789-3905","authenticated-orcid":false,"given":"Banita","family":"Yadav","sequence":"additional","affiliation":[{"name":"BRIC\u2010National Institute of Plant Genome Research (NIPGR)  New Delhi India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abhilash","family":"Dasari","sequence":"additional","affiliation":[{"name":"Indian Institute of Technology  New Delhi India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1702-2516","authenticated-orcid":false,"given":"Rushil","family":"Mandlik","sequence":"additional","affiliation":[{"name":"Central University of Haryana  Mahendergarh Haryana India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4796-6120","authenticated-orcid":false,"given":"Humira","family":"Sonah","sequence":"additional","affiliation":[{"name":"Central University of Haryana  Mahendergarh Haryana India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4167-6552","authenticated-orcid":false,"given":"Rupesh","family":"Deshmukh","sequence":"additional","affiliation":[{"name":"Central University of Haryana  Mahendergarh Haryana India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ishaan","family":"Gupta","sequence":"additional","affiliation":[{"name":"Indian Institute of Technology  New Delhi India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0404-4532","authenticated-orcid":false,"given":"Petra","family":"Bauer","sequence":"additional","affiliation":[{"name":"Institute of Botany Heinrich Heine University  D\u00fcsseldorf Germany"},{"name":"Cluster of Excellence on Plant Science (CEPLAS), Heinrich\u2010Heine\u2010University  D\u00fcsseldorf Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0893-3895","authenticated-orcid":false,"given":"Hasthi","family":"Ram","sequence":"additional","affiliation":[{"name":"BRIC\u2010National Institute of Plant Genome Research (NIPGR)  New Delhi India"},{"name":"Institute of Botany Heinrich Heine University  D\u00fcsseldorf Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,4]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"crossref","first-page":"721","DOI":"10.1111\/pbi.13239","article-title":"Generation of a Multiplex Mutagenesis Population via Pooled CRISPR\u2010Cas9 in Soya Bean","volume":"18","author":"Bai M.","year":"2020","journal-title":"Plant Biotechnology Journal"},{"key":"e_1_2_8_3_1","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1007\/s004250050545","article-title":"Structure and Regulation of OPR1 and OPR2, Two Closely Related Genes Encoding 12\u2010Oxophytodienoic Acid\u201010,11\u2010Reductases From \n                     Arabidopsis thaliana","volume":"208","author":"Biesgen C.","year":"1999","journal-title":"Planta"},{"key":"e_1_2_8_4_1","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1089\/crispr.2020.0022","article-title":"Deconvolution of Complex DNA Repair (DECODR): Establishing a Novel Deconvolution Algorithm for Comprehensive Analysis of CRISPR\u2010Edited Sanger Sequencing Data","volume":"4","author":"Bloh K.","year":"2021","journal-title":"CRISPR Journal"},{"key":"e_1_2_8_5_1","doi-asserted-by":"crossref","DOI":"10.3390\/plants15091312","article-title":"Differential Jasmonate Profiles in Oat Roots and Leaves Reveal a Role for 12\u2010Oxo Phytodienoic Acid (OPDA) in Drought Tolerance by Modulating Root Growth","volume":"15","author":"Canales F. J.","year":"2026","journal-title":"Plants"},{"key":"e_1_2_8_6_1","doi-asserted-by":"crossref","first-page":"770","DOI":"10.1104\/pp.111.174169","article-title":"Intronic T\u2010DNA Insertion Renders Arabidopsis opr3 a Conditional Jasmonic Acid\u2010Producing Mutant","volume":"156","author":"Chehab E. W.","year":"2011","journal-title":"Plant Physiology"},{"key":"e_1_2_8_7_1","doi-asserted-by":"crossref","DOI":"10.1038\/s41467-023-36248-y","article-title":"Dosage Differences in 12\u2010OXOPHYTODIENOATE REDUCTASE Genes Modulate Wheat Root Growth","volume":"14","author":"Gabay G.","year":"2023","journal-title":"Nature Communications"},{"key":"e_1_2_8_8_1","doi-asserted-by":"crossref","first-page":"2023","DOI":"10.1104\/pp.17.00489","article-title":"Generation of a Collection of Mutant Tomato Lines Using Pooled CRISPR Libraries","volume":"174","author":"Jacobs T. B.","year":"2017","journal-title":"Plant Physiology"},{"key":"e_1_2_8_9_1","doi-asserted-by":"crossref","DOI":"10.1002\/pei3.70133","article-title":"CRISPR\/Cas Genome Editing and Its Applications in Cereal Crop Improvement","volume":"7","author":"Kaniganti S.","year":"2026","journal-title":"Plant\u2010Environment Interactions"},{"key":"e_1_2_8_10_1","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1046\/j.1365-313X.2003.01948.x","article-title":"Establishing an Efficient Ac\/Ds Tagging System in Rice: Large\u2010Scale Analysis of ds Flanking Sequences","volume":"37","author":"Kolesnik T.","year":"2004","journal-title":"Plant Journal"},{"key":"e_1_2_8_11_1","doi-asserted-by":"crossref","first-page":"1494","DOI":"10.1093\/mp\/ssu044","article-title":"CRISPR\u2010P: A Web Tool for Synthetic Single\u2010Guide RNA Design of CRISPR\u2010System in Plants","volume":"7","author":"Lei Y.","year":"2014","journal-title":"Molecular Plant"},{"key":"e_1_2_8_12_1","doi-asserted-by":"crossref","first-page":"981","DOI":"10.1007\/s00299-011-1002-5","article-title":"Comparative Characterization, Expression Pattern and Function Analysis of the 12\u2010Oxo\u2010Phytodienoic Acid Reductase Gene Family in Rice","volume":"30","author":"Li W.","year":"2011","journal-title":"Plant Cell Reports"},{"key":"e_1_2_8_13_1","doi-asserted-by":"crossref","first-page":"1397","DOI":"10.1105\/tpc.19.00934","article-title":"High\u2010Throughput CRISPR\/Cas9 Mutagenesis Streamlines Trait Gene Identification in Maize","volume":"32","author":"Liu H.\u2010J.","year":"2020","journal-title":"Plant Cell"},{"key":"e_1_2_8_14_1","doi-asserted-by":"crossref","first-page":"1242","DOI":"10.1016\/j.molp.2017.06.007","article-title":"Genome\u2010Wide Targeted Mutagenesis in Rice Using the CRISPR\/Cas9 System","volume":"10","author":"Lu Y.","year":"2017","journal-title":"Molecular Plant"},{"key":"e_1_2_8_15_1","doi-asserted-by":"crossref","first-page":"11541","DOI":"10.1073\/pnas.1831119100","article-title":"Maize\u2010Targeted Mutagenesis: A Knockout Resource for Maize","volume":"100","author":"May B. P.","year":"2003","journal-title":"Proceedings of the National Academy of Sciences of the United States of America"},{"key":"e_1_2_8_16_1","doi-asserted-by":"crossref","first-page":"1238","DOI":"10.1016\/j.molp.2017.06.006","article-title":"Construction of a Genome\u2010Wide Mutant Library in Rice Using CRISPR\/Cas9","volume":"10","author":"Meng X.","year":"2017","journal-title":"Molecular Plant"},{"key":"e_1_2_8_17_1","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1038\/nrg730","article-title":"The Art and Design of Genetic Screens: \n                     Arabidopsis thaliana","volume":"3","author":"Page D. R.","year":"2002","journal-title":"Nature Reviews Genetics"},{"key":"e_1_2_8_18_1","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1007\/s00709-023-01925-8","article-title":"A Simplified and Improved Protocol of Rice Transformation to Cater Wide Range of Rice Cultivars","volume":"261","author":"Rengasamy B.","year":"2024","journal-title":"Protoplasma"},{"key":"e_1_2_8_19_1","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1002\/0471142727.mb0203s27","article-title":"Preparation of Genomic DNA From Plant Tissue","volume":"27","author":"Richards E.","year":"1994","journal-title":"CP Molecular Biology"},{"key":"e_1_2_8_20_1","doi-asserted-by":"crossref","first-page":"979","DOI":"10.1007\/s004250050706","article-title":"12\u2010Oxophytodienoate Reductase 3 (OPR3) is the Isoenzyme Involved in Jasmonate Biosynthesis","volume":"210","author":"Schaller F.","year":"2000","journal-title":"Planta"},{"key":"e_1_2_8_21_1","doi-asserted-by":"crossref","DOI":"10.1093\/aobpla\/plab056","article-title":"RhizoVision Explorer: Open\u2010Source Software for Root Image Analysis and Measurement Standardization","volume":"13","author":"Seethepalli A.","year":"2021","journal-title":"AoB Plants"},{"key":"e_1_2_8_22_1","doi-asserted-by":"crossref","first-page":"517","DOI":"10.1007\/s00425-007-0635-7","article-title":"Identification of the OsOPR7 Gene Encoding 12\u2010Oxophytodienoate Reductase Involved in the Biosynthesis of Jasmonic Acid in Rice","volume":"227","author":"Tani T.","year":"2008","journal-title":"Planta"},{"issue":"1","key":"e_1_2_8_23_1","doi-asserted-by":"crossref","first-page":"198","DOI":"10.1007\/s11033-023-09159-w","article-title":"Functional Analysis of a Rice 12\u2010Oxo\u2010Phytodienoic Acid Reductase Gene (OsOPR1) Involved in cd Stress Tolerance","volume":"51","author":"Wu L.","year":"2024","journal-title":"Molecular Biology Reports"},{"key":"e_1_2_8_24_1","doi-asserted-by":"crossref","first-page":"3570","DOI":"10.1073\/pnas.1420294112","article-title":"Boosting CRISPR\/Cas9 Multiplex Editing Capability With the Endogenous tRNA\u2010Processing System","volume":"112","author":"Xie K.","year":"2015","journal-title":"Proceedings of the National Academy of Sciences of the United States of America"},{"key":"e_1_2_8_25_1","doi-asserted-by":"crossref","first-page":"1769","DOI":"10.1186\/s12870-025-07769-z","article-title":"A CRISPR\u2010Cas9 Library to Target Putative Redundant Gene Sets Facilitates Their Functional Exploration in Grain Development in Rice","volume":"25","author":"Yadav B.","year":"2025","journal-title":"BMC Plant Biology"},{"key":"e_1_2_8_26_1","doi-asserted-by":"crossref","DOI":"10.3390\/mps6060113","article-title":"A Versatile Protocol for Efficient Transformation and Regeneration in Mega Indica Rice Cultivar MTU1010: Optimization Through Hormonal Variables","volume":"6","author":"Yadav P.","year":"2023","journal-title":"Methods and Protocols"},{"key":"e_1_2_8_27_1","volume-title":"Laboratory Manual for Physiological Studies of Rice","author":"Yoshida S.","year":"1971"}],"container-title":["Physiologia Plantarum"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/ppl.71097","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1111\/ppl.71097","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/ppl.71097","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T09:42:00Z","timestamp":1788514920000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1111\/ppl.71097"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9]]},"references-count":26,"journal-issue":{"issue":"5","published-print":{"date-parts":[[2026,9]]}},"alternative-id":["10.1111\/ppl.71097"],"URL":"https:\/\/doi.org\/10.1111\/ppl.71097","archive":["Portico"],"relation":{},"ISSN":["0031-9317","1399-3054"],"issn-type":[{"value":"0031-9317","type":"print"},{"value":"1399-3054","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9]]},"assertion":[{"value":"2026-06-17","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-08-26","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-09-04","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e71097"}}