Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Biogenesis of Lysosome-related Organelle Mutant Silkworms by Direct Injection of a Cas9 Protein-guided RNA Complex into Bombyx mori Embryos

Cas9 단백질/ 가이드 RNA 복합체를 이용한 누에 BmBLOS 유전자 편집

  • Kim, Kee Young (Sericultural and Apicultural Materials Division, National Institute of Agricultural Science, RDA) ;
  • Yu, Jeong Hee (Sericultural and Apicultural Materials Division, National Institute of Agricultural Science, RDA) ;
  • Kim, Su-Bae (Sericultural and Apicultural Materials Division, National Institute of Agricultural Science, RDA) ;
  • Kim, Seong-Wan (Sericultural and Apicultural Materials Division, National Institute of Agricultural Science, RDA) ;
  • Kim, Seong-Ryul (Sericultural and Apicultural Materials Division, National Institute of Agricultural Science, RDA) ;
  • Choi, Kwang-Ho (Sericultural and Apicultural Materials Division, National Institute of Agricultural Science, RDA) ;
  • Kim, Jong Gil (Sericultural and Apicultural Materials Division, National Institute of Agricultural Science, RDA) ;
  • Park, Jong Woo (Sericultural and Apicultural Materials Division, National Institute of Agricultural Science, RDA)
  • 김기영 (국립농업과학원 잠사양봉소재과) ;
  • 유정희 (국립농업과학원 잠사양봉소재과) ;
  • 김수배 (국립농업과학원 잠사양봉소재과) ;
  • 김성완 (국립농업과학원 잠사양봉소재과) ;
  • 김성렬 (국립농업과학원 잠사양봉소재과) ;
  • 최광호 (국립농업과학원 잠사양봉소재과) ;
  • 김종길 (국립농업과학원 잠사양봉소재과) ;
  • 박종우 (국립농업과학원 잠사양봉소재과)
  • Received : 2019.03.26
  • Accepted : 2019.04.16
  • Published : 2019.05.30
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Abstract

Genome editing technology employing gene scissors has generated interest in molecular breeding in various fields, and the development of the third-generation gene scissors of the clustered, regularly interspaced short palindromic repeat (CRISPR) system has accelerated the field of molecular breeding through genome editing. In this study, we analyzed the possibility of silkworm molecular breeding using gene scissors by genomic and phenotypic analysis after editing the biogenesis of lysosome-related organelles (BmBLOS) gene of Bakokjam using the CRISPR/Cas9 system. Three types of guide RNAs (gRNA) were synthesized based on the BmBLOS gene sequence of Bakokjam. Complexes of the prepared gRNA and Cas9 protein were formed and introduced into Bombyx mori BM-N cells by electroporation. Analysis of the gene editing efficiency by T7 endonuclease I analysis revealed that the B4N gRNA showed the best efficiency. The silkworm genome was edited by microinjecting the Cas9/B4N gRNA complex into silkworm early embryos and raising the silkworms after hatching. The hatching rate was as low as 18%, but the incidence of mutation was over 40%. In addition, phenotypic changes were observed in about 70% of the G0 generation silkworms. Sequence analysis showed that the BmBLOS gene appeared to be a heterozygote carrying the wild-type and mutation in most individuals, and the genotype of the BmBLOS gene was also different in all individuals. These results suggest that although the possibility of silkworm molecular breeding using the CRISPR/Cas9 system would be very high, continued research on breeding and screening methods will be necessary to improve gene editing efficiency and to obtain homozygotes.

유전자 가위를 이용한 게놈편집 기술의 등장은 다양한 분야에서 분자육종에 대한 관심을 유발하였으며, 3세대 유전자가위 CRISPR 기술의 개발은 게놈편집을 통한 분자육종 시대를 가속화하고 있다. 본 연구에서는 최근 개발된 3세대 유전자 가위 CRISPR/Cas9을 이용하여 국내 보급품종인 백옥잠의 BmBLOS 유전자를 편집하여 돌연변이를 유도하고 유전형 및 표현형 검사를 통하여 유전자가위를 이용한 누에 분자육종가능성을 분석하고 이용기술을 확보하고자 하였다. 유전자 편집을 위하여 백옥잠의 BmBLOS 유전자의 염기서열을 구명하고, 이를 바탕으로 3종의 가이드 RNA를 합성하였다. 합성된 gRNA는 Cas9 단백질과 복합체를 형성시킨 후 BM-N 누에 세포주에 도입 후 T7 endonuclease I 분석을 통하여 편집효율이 가장 높은 B4N gRNA를 선발하였다. 누에 유전자를 편집하기 위하여 Cas9/B4N gRNA를 누에 초가 배아에 미세주사하고 사육하였다. 미세주사 후 부화율은 18% 가량으로 낮게 나타났으나 생존한 개체 중 돌연변이 발생율은 40% 이상으로 비교적 높게 나타났다. 또한 유전자 편집 G0 세대누에 중 70% 가량에서 표현형의 변화가 관찰되었고, 염기서열 분석결과 대부분의 개체에서 BmBLOS 유전자가 정상과 돌연변이가 같이 존재하는 이형접합자 형태로 나타났으며, 그 유전형 또한 모든 개체에서 다르게 나타났다. 이러한 결과에 비추어 볼 때 CRISPR/Cas9 시스템을 이용한 누에 분자육종의 가능성은 매우 높을 것으로 예상되나, 유전자 편집효율을 개선하고 동형접합자를 얻기 위한 교배 및 선발방법에 대한 지속적인 연구가 필요하다고 판단된다.

Keywords

SMGHBM_2019_v29n5_537_f0001.png 이미지

Fig. 1. Design and delivery of gRNA.

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Fig. 2. Gene editing in the B. mori genome.

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Fig. 4. Relative gene expression of in BmBLOS mutants.

SMGHBM_2019_v29n5_537_f0005.png 이미지

Fig. 3. Mutations of BmBLOS gene induced by Cas9/B4N gRNA injection.

Table 1. Primers used in this study

SMGHBM_2019_v29n5_537_t0001.png 이미지

Table 2. Analysis of gRNA target sequences of the BmBLOS gene and potential off-target in the genome

SMGHBM_2019_v29n5_537_t0002.png 이미지

Table 3. Embryonic gene editing induced by Cas9/gRNA complex injection targeting BmBLOS

SMGHBM_2019_v29n5_537_t0003.png 이미지

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