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Genome editing of hybrid poplar (Populus alba × P. glandulosa) protoplasts using Cas9/gRNA ribonucleoprotein
J Plant Biotechnol 2021;48:34-43
Published online March 31, 2021
© 2021 The Korean Society for Plant Biotechnology.

Su Jin Park ·Young-Im Choi ·Hyun A Jang ·Sang-Gyu Kim·Hyunmo Choi ·Beum-Chang Kang ·Hyoshin Lee ·Eun-Kyung Bae

Department of Forest Bio-resources, National Institute of Forest Science, Suwon 16631, Korea
Department of Biological Science, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea
Forest Biomaterials Research Center, National Institute of Forest Science, Jinju 52817, Korea)
Center for Genome Engineering, Institute for Basic Science (IBS), Daejeon 34126, Korea
Correspondence to: e-mail: baeek@korea.kr
Received November 23, 2020; Revised January 5, 2021; Accepted January 5, 2021.
cc This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Targeted genome editing using the CRISPR/Cas9 system is a ground-breaking technology that is being widely used to produce plants with useful traits. However, for woody plants, only a few successful attempts have been reported. These successes have used Agrobacterium-mediated transformation, which has been reported to be very efficient at producing genetically modified trees. Nonetheless, there are unresolved problems with plasmid sequences that remain in the plant genome. In this study, we demonstrated a DNA-free genome editing technique in which purified CRISPR/Cas9 ribonucleoproteins (RNPs) are delivered directly to the protoplasts of a hybrid poplar (Populus alba × Populus glandulosa). We designed three single-guide RNAs (sgRNAs) to target the stress-associated protein 1 gene (PagSAP1) in the hybrid poplar. Deep sequencing results showed that pre-assembled RNPs had a more efficient target mutagenesis insertion and deletion (indel) frequency than did non-assembled RNPs. Moreover, the RNP of sgRNA3 had a significantly higher editing efficacy than those of sgRNA1 and sgRNA2. Our results suggest that the CRISPR/Cas9 ribonucleoproteinmediated transfection approach is useful for the production of transgene-free genome-edited tree plants.
Keywords : CRISPR/Cas9, Hybrid poplar, PagSAP1, Protoplast, Ribonucleoproteins


March 2021, 48 (1)
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