PMID- 38003326 OWN - NLM STAT- MEDLINE DCOM- 20231127 LR - 20231127 IS - 1422-0067 (Electronic) IS - 1422-0067 (Linking) VI - 24 IP - 22 DP - 2023 Nov 9 TI - CRISPR/Cas9 Mutagenesis through Introducing a Nanoparticle Complex Made of a Cationic Polymer and Nucleic Acids into Maize Protoplasts. LID - 10.3390/ijms242216137 [doi] LID - 16137 AB - Presently, targeted gene mutagenesis attracts increasing attention both in plant research and crop improvement. In these approaches, successes are largely dependent on the efficiency of the delivery of gene editing components into plant cells. Here, we report the optimization of the cationic polymer poly(2-hydroxypropylene imine) (PHPI)-mediated delivery of plasmid DNAs, or single-stranded oligonucleotides labelled with Cyanine3 (Cy3) or 6-Carboxyfluorescein (6-FAM)-fluorescent dyes into maize protoplasts. Co-delivery of the GFP-expressing plasmid and the Cy3-conjugated oligonucleotides has resulted in the cytoplasmic and nuclear accumulation of the green fluorescent protein and a preferential nuclear localization of oligonucleotides. We show the application of nanoparticle complexes, i.e., "polyplexes" that comprise cationic polymers and nucleic acids, for CRISPR/Cas9 editing of maize cells. Knocking out the functional EGFP gene in transgenic maize protoplasts was achieved through the co-delivery of plasmids encoding components of the editing factors Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) after complexing with a cationic polymer (PHPI). Several edited microcalli were identified based on the lack of a GFP fluorescence signal. Multi-base and single-base deletions in the EGFP gene were confirmed using Sanger sequencing. The presented results support the use of the PHPI cationic polymer in plant protoplast-mediated genome editing approaches. FAU - Nagy, Bettina AU - Nagy B AD - Agribiotechnology and Precision Breeding for Food Security National Laboratory, Institute of Plant Biology, HUN-REN Biological Research Centre, 6726 Szeged, Hungary. FAU - Oktem, Aysegul AU - Oktem A AUID- ORCID: 0000-0001-7828-999X AD - Laboratory of Cellular Imaging, Core Facilities, HUN-REN Biological Research Centre, 6726 Szeged, Hungary. AD - Department of Medical Microbiology, University Medical Center, University of Groningen, 9700 Groningen, The Netherlands. FAU - Ferenc, Gyorgyi AU - Ferenc G AUID- ORCID: 0000-0002-3456-319X AD - Agribiotechnology and Precision Breeding for Food Security National Laboratory, Institute of Plant Biology, HUN-REN Biological Research Centre, 6726 Szeged, Hungary. FAU - Ungor, Ditta AU - Ungor D AUID- ORCID: 0000-0002-7659-0428 AD - Department of Physical Chemistry and Materials Science, University of Szeged, 6720 Szeged, Hungary. FAU - Kalac, Aladina AU - Kalac A AD - Laboratory of Cellular Imaging, Core Facilities, HUN-REN Biological Research Centre, 6726 Szeged, Hungary. FAU - Kelemen-Valkony, Ildiko AU - Kelemen-Valkony I AD - Laboratory of Cellular Imaging, Core Facilities, HUN-REN Biological Research Centre, 6726 Szeged, Hungary. FAU - Fodor, Elfrieda AU - Fodor E AUID- ORCID: 0000-0002-5695-6162 AD - Institute of Biochemistry, HUN-REN Biological Research Centre, 6726 Szeged, Hungary. FAU - Nagy, Istvan AU - Nagy I AD - Institute of Biochemistry, HUN-REN Biological Research Centre, 6726 Szeged, Hungary. AD - SeqOmics Biotechnology Ltd., 6782 Morahalom, Hungary. FAU - Dudits, Denes AU - Dudits D AD - Agribiotechnology and Precision Breeding for Food Security National Laboratory, Institute of Plant Biology, HUN-REN Biological Research Centre, 6726 Szeged, Hungary. FAU - Ayaydin, Ferhan AU - Ayaydin F AD - Agribiotechnology and Precision Breeding for Food Security National Laboratory, Institute of Plant Biology, HUN-REN Biological Research Centre, 6726 Szeged, Hungary. AD - Laboratory of Cellular Imaging, Core Facilities, HUN-REN Biological Research Centre, 6726 Szeged, Hungary. AD - Functional Cell Biology and Immunology Advanced Core Facility (FCBI), Hungarian Centre of Excellence for Molecular Medicine (HCEMM), 6720 Szeged, Hungary. AD - Faculty of Medicine, Albert Szent-Gyorgyi Health Centre, Interdisciplinary R&D and Innovation Centre of Excellence, University of Szeged, 6725 Szeged, Hungary. LA - eng GR - RRF-2.3.1-21-2022-00007/National Research, Development and Innovation Office of the Hungarian Government/ GR - 739593/European Union's Horizon 2020 research and innovation program/ PT - Journal Article DEP - 20231109 PL - Switzerland TA - Int J Mol Sci JT - International journal of molecular sciences JID - 101092791 RN - 0 (Nucleic Acids) RN - 0 (Polymers) RN - 0 (RNA, Guide, CRISPR-Cas Systems) RN - 147336-22-9 (Green Fluorescent Proteins) RN - 0 (Oligonucleotides) SB - IM MH - CRISPR-Cas Systems/genetics MH - Protoplasts MH - Zea mays/genetics MH - *Nucleic Acids MH - Polymers MH - RNA, Guide, CRISPR-Cas Systems MH - Mutagenesis MH - Gene Editing/methods MH - Green Fluorescent Proteins/genetics MH - Oligonucleotides MH - *Nanoparticles PMC - PMC10671792 OTO - NOTNLM OT - CRISPR/Cas9 OT - DNA oligonucleotide OT - DsRed fluorescent marker protein OT - GFP expression OT - maize protoplasts OT - mutagenesis OT - nanoparticle OT - plasmid uptake OT - poly (2-hydroxypropylene imine) OT - polyplexes COIS- The authors declare no conflict of interest. Author I. Nagy was employed by the company SeqOmics Biotechnology Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. EDAT- 2023/11/25 12:43 MHDA- 2023/11/27 16:18 PMCR- 2023/11/09 CRDT- 2023/11/25 01:15 PHST- 2023/10/04 00:00 [received] PHST- 2023/10/31 00:00 [revised] PHST- 2023/11/06 00:00 [accepted] PHST- 2023/11/27 16:18 [medline] PHST- 2023/11/25 12:43 [pubmed] PHST- 2023/11/25 01:15 [entrez] PHST- 2023/11/09 00:00 [pmc-release] AID - ijms242216137 [pii] AID - ijms-24-16137 [pii] AID - 10.3390/ijms242216137 [doi] PST - epublish SO - Int J Mol Sci. 2023 Nov 9;24(22):16137. doi: 10.3390/ijms242216137.