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http://dx.doi.org/10.5010/JPB.2021.48.1.034

Genome editing of hybrid poplar (Populus alba × P. glandulosa) protoplasts using Cas9/gRNA ribonucleoprotein  

Park, Su Jin (Department of Forest Bio-resources, National Institute of Forest Science)
Choi, Young-Im (Department of Forest Bio-resources, National Institute of Forest Science)
Jang, Hyun A (Department of Forest Bio-resources, National Institute of Forest Science)
Kim, Sang-Gyu (Department of Biological Science, Korea Advanced Institute of Science and Technology (KAIST))
Choi, Hyunmo (Forest Biomaterials Research Center, National Institute of Forest Science)
Kang, Beum-Chang (Center for Genome Engineering, Institute for Basic Science (IBS))
Lee, Hyoshin (Department of Forest Bio-resources, National Institute of Forest Science)
Bae, Eun-Kyung (Department of Forest Bio-resources, National Institute of Forest Science)
Publication Information
Journal of Plant Biotechnology / v.48, no.1, 2021 , pp. 34-43 More about this Journal
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 ribonucleoprotein-mediated transfection approach is useful for the production of transgene-free genome-edited tree plants.
Keywords
CRISPR/Cas9; Hybrid poplar; PagSAP1; Protoplast; Ribonucleoproteins;
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