PMID- 31360827 OWN - NLM STAT- PubMed-not-MEDLINE LR - 20231013 IS - 2475-4455 (Electronic) IS - 2475-4455 (Linking) VI - 3 IP - 7 DP - 2019 Jul TI - Targeted insertion of large DNA sequences by homology-directed repair or non-homologous end joining in engineered tobacco BY-2 cells using designed zinc finger nucleases. PG - e00153 LID - 10.1002/pld3.153 [doi] LID - e00153 AB - Targeted integration of recombinant DNA fragments into plant genomes by DNA double-strand break (DSB) repair mechanisms has become a powerful tool for precision engineering of crops. However, many targeting platforms require the screening of many transgenic events to identify a low number of targeted events among many more random insertion events. We developed an engineered transgene integration platform (ETIP) that uses incomplete marker genes at the insertion site to enable rapid phenotypic screening and recovery of targeted events upon functional reconstitution of the marker genes. The two marker genes, encoding neomycin phosphotransferase II (nptII) and Discosoma sp. red fluorescent protein (DsRed) enable event selection on kanamycin-containing selective medium and subsequent screening for red fluorescent clones. The ETIP design allows targeted integration of donor DNA molecules either by homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated mechanisms. Targeted donor DNA integration is facilitated by zinc finger nucleases (ZFN). The ETIP cassette was introduced into Nicotiana tabacum BY-2 suspension cells to generate target cell lines containing a single copy locus of the transgene construct. The utility of the ETIP platform has been demonstrated by targeting DNA constructs containing up to 25-kb payload. The success rate for clean targeted DNA integration was up to 21% for HDR and up to 41% for NHEJ based on the total number of calli analyzed by next-generation sequencing (NGS). The rapid generation of targeted events with large DNA constructs expands the utility of the nuclease-mediated gene addition platform both for academia and the commercial sector. FAU - Schiermeyer, Andreas AU - Schiermeyer A AD - Fraunhofer Institute for Molecular Biology and Applied Ecology IME Aachen Germany. FAU - Schneider, Katja AU - Schneider K AD - Fraunhofer Institute for Molecular Biology and Applied Ecology IME Aachen Germany. FAU - Kirchhoff, Janina AU - Kirchhoff J AD - Fraunhofer Institute for Molecular Biology and Applied Ecology IME Aachen Germany. FAU - Schmelter, Thomas AU - Schmelter T AD - Fraunhofer Institute for Molecular Biology and Applied Ecology IME Aachen Germany. FAU - Koch, Natalie AU - Koch N AD - Fraunhofer Institute for Molecular Biology and Applied Ecology IME Aachen Germany. FAU - Jiang, Ke AU - Jiang K AD - Corteva Agriscience Indianapolis IN USA. FAU - Herwartz, Denise AU - Herwartz D AD - Fraunhofer Institute for Molecular Biology and Applied Ecology IME Aachen Germany. FAU - Blue, Ryan AU - Blue R AD - Corteva Agriscience Indianapolis IN USA. FAU - Marri, Pradeep AU - Marri P AD - Corteva Agriscience Indianapolis IN USA. FAU - Samuel, Pon AU - Samuel P AD - Corteva Agriscience Indianapolis IN USA. FAU - Corbin, David R AU - Corbin DR AD - Corteva Agriscience Indianapolis IN USA. FAU - Webb, Steven R AU - Webb SR AD - Corteva Agriscience Indianapolis IN USA. FAU - Gonzalez, Delkin O AU - Gonzalez DO AD - Corteva Agriscience Indianapolis IN USA. FAU - Folkerts, Otto AU - Folkerts O AD - Corteva Agriscience Indianapolis IN USA. FAU - Fischer, Rainer AU - Fischer R AD - Fraunhofer Institute for Molecular Biology and Applied Ecology IME Aachen Germany. AD - Indiana Biosciences Research Institute Indianapolis IN USA. FAU - Schinkel, Helga AU - Schinkel H AD - Fraunhofer Institute for Molecular Biology and Applied Ecology IME Aachen Germany. FAU - Ainley, W Michael AU - Ainley WM AD - Corteva Agriscience Indianapolis IN USA. FAU - Schillberg, Stefan AU - Schillberg S AD - Fraunhofer Institute for Molecular Biology and Applied Ecology IME Aachen Germany. LA - eng PT - Journal Article DEP - 20190719 PL - England TA - Plant Direct JT - Plant direct JID - 101716131 PMC - PMC6639735 OTO - NOTNLM OT - DNA recombination OT - electroporation OT - gene targeting OT - genome editing OT - particle bombardment OT - split marker genes OT - zinc finger nucleases COIS- K.J., R.B., P.M., P.S., D.R.C, S.R.W., D.O.G., W.M.A., and O.F. were employed by Dow AgroSciences LLC, a wholly owned subsidiary of The Dow Chemical Company, at the time the work was performed, and Dow AgroSciences provided funding for the research. Dow AgroSciences is now part of Corteva Agrisciences, which has filed a patent application on the ETIP concept and the cell lines containing the targeting cassette. All authors declare no conflict of interest. EDAT- 2019/07/31 06:00 MHDA- 2019/07/31 06:01 PMCR- 2019/07/19 CRDT- 2019/07/31 06:00 PHST- 2019/04/10 00:00 [received] PHST- 2019/06/11 00:00 [revised] PHST- 2019/07/03 00:00 [accepted] PHST- 2019/07/31 06:00 [entrez] PHST- 2019/07/31 06:00 [pubmed] PHST- 2019/07/31 06:01 [medline] PHST- 2019/07/19 00:00 [pmc-release] AID - PLD3153 [pii] AID - 10.1002/pld3.153 [doi] PST - epublish SO - Plant Direct. 2019 Jul 19;3(7):e00153. doi: 10.1002/pld3.153. eCollection 2019 Jul.