Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Genome editing of hybrid poplar (Populus alba × P. glandulosa) protoplasts using Cas9/gRNA ribonucleoprotein

현사시나무 원형질체에서 리보핵산단백질을 활용한 유전자 교정 방법 연구

  • 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)
  • 박수진 (국립산림과학원 산림생명자원연구부) ;
  • 최영임 (국립산림과학원 산림생명자원연구부) ;
  • 장현아 (국립산림과학원 산림생명자원연구부) ;
  • 김상규 (한국과학기술원 생명과학과) ;
  • 최현모 (국립산림과학원 산림바이오소재연구소) ;
  • 강범창 (기초과학연구원 유전체 교정 연구단) ;
  • 이효신 (국립산림과학원 산림생명자원연구부) ;
  • 배은경 (국립산림과학원 산림생명자원연구부)
  • Received : 2020.11.23
  • Accepted : 2021.01.05
  • Published : 2021.03.31
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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.

CRISPR/Cas9에 의한 유전자 교정 기술은 유용 형질을 갖는 작물 및 임목의 육성에 있어 널리 사용되고 있는 핵심 기술이다. 유전자 교정 임목 육성에는 아그로박테리움에 의한 형질전환 방법이 높은 효율로 시행된 연구가 많았고 따라서 형질전환에 사용된 플라스미드 서열이 식물 유전체 안에 존재한다는 문제가 남아 있었다. 본 연구에서는 CRISPR/Cas9을 사용하여 유전자 교정 임목을 육성하는 데 기존에 알려진 벡터 도입 기술이 아닌, 단일 가닥 가이드 RNA (sgRNA)와 Cas9 단백질을 혼합하여 만든 리보핵산단백질을 현사시나무 원형질체에 도입하는 방법을 기술하였다. 염 스트레스 내성 관련 인자 PagSAP1 유전자를 표적으로 하는 3종류의 sgRNA를 디자인하고, 각 sgRNA와 Cas9 단백질을 혼합하여 만든 리보핵산단백질을 원형질체에 도입하였다. 표적화 딥시퀀싱을 통해 리보핵산단백질 형성 시 sgRNA와 Cas9 단백질을 혼합하고 일정 시간 배양하여 안정화되는 시간이 필요한 것을 확인하였다. 또한 sgRNA3의 리보핵산단백질이 sgRNA1, sgRNA2의 리보핵산단백질보다 높은 교정 효율을 보이는 것을 확인하였다. 본 실험을 통해 리보핵산단백질을 이용한 유전자 교정 기술이 임목에도 적용될 수 있음이 확인되었고, 이는 외래 유전자 없이 유전자 교정 임목을 육성하는 데 활용할 수 있을 것으로 사료된다.

Keywords

Acknowledgement

본 연구는 산림청 국립산림과학원 연구사업(세부과제명: 유전자가위를 이용한 임목 유전체 교정 기술 개발, 과제번호: FG0702-2018-01-2018)의 지원에 의해 이루어진 것임.

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