[논문]CRISPR/Cas9 System을 활용한 배스의 불임 유도에 대한 연구

박승철; 김종현; 이윤정

doi:10.13047/kjee.2021.35.5.503

CRISPR/Cas9 System을 활용한 배스의 불임 유도에 대한 연구
A Study on the Induction of Infertility of Largemouth Bass (Micropterus salmoides) by CRISPR/Cas9 System 원문보기

한국환경생태학회지 = Korean journal of environment and ecology, v.35 no.5, 2021년, pp.503 - 524

박승철 (국립공원연구원) , 김종현 (주식회사 엔에이피) , 이윤정 (주식회사 엔에이피)

초록
AI-Helper

배스(Micropterus salmoides)는 수생태계에서 최상위단계에 위치하는 생태계교란 어종으로 심각한 담수생태계의 불균형을 초래하고 있다. 배스의 퇴치 및 관리를 위한 다양한 시도를 하고 있지만 효과적인 방안은 없는 상황이므로 배스의 고유한 특성에 기반한 개체군 감소의 효율성을 극대화할 수 있는 방식을 모색하였다. 본 연구에서는 배스의 Transcriptom 분석으로 Unigene contigs는 182,887개, 그리고 정자-난자 인식 단백질인 IZUMO1과 Zona pellucida sperm-binding protein의 유전자에서 CRISPR/Cas9 system을 적용할 최종 Target sequence는 12종을 산출하였다. 각 Target sequence를 인식할 수 있는 12종의 sgRNA를 합성한 후 후속 연구에 사용할 12종의 Cas9-sgRNA ribonucleoprotein (RNP) complex를 제작하였다. 본 연구에서는 차세대염기서열 분석법으로 정자-난자 인식 단백질을 암호화하는 유전자를 탐색하였고, CRISPR/Cas9 system으로 유전자를 편집하여 번식행동은 하지만 수정란을 형성하지 못하는 생식세포를 생산하는 불임개체를 유도하기 위한 조성물 개발 과정을 확립하였다. 그리고 배스와 동일한 수계에 있는 고유 생물종의 서식에는 영향을 미치지 않는 생태교란종 관리 방안으로서의 유용성을 검증하기 위한 후속 연구의 귀중한 기초 자료를 확보하는데 기여했다고 판단된다.

Abstract ▼ AI-Helper

A largemouth bass (Micropterus salmoides) is an ecosystem disturbance fish species at the highest rank in the aquatic ecosystem, causing a serious imbalance in freshwater ecosystems. Although various attempts have been made to eradicate and control largemouth bass, no effective measures were found. Therefore, it is necessary to find an approach to maximize the effective population reduction based on the unique characteristics of largemouth bass. This study used the transcriptome analysis to derive 182,887 unigene contigs and select 12 types of final target sequences for applying the CRISPR/Cas9 system in the genes of IZUMO1 and Zona pellucida sperm-binding protein, which are proteins involved in sperm-egg recognition. After synthesizing 12 types of sgRNA capable of recognizing each target sequence, 12 types of Cas9-sgRNA ribonucleoprotein (RNP) complexes to be used in subsequent studies were prepared. This study searched the protein-coding gene of sperm-egg through the Next Generation Sequencing (NGS) and edited genes through the CRISPR/Cas9 system to induce infertile individuals that produced reproductive cells but could not form fertilized eggs. Through such a series of processes, it successfully established a composition development process for largemouth bass. It is judged that this study contributed to securing the valuable basic data for follow-up studies to verify its effect for the management of ecological disturbances without affecting the habitat of other endemic species in the same water system with the largemouth bass.

주제어

표/그림 (24)

그림 Figure 1. Flow Chart for the Development Process of infertile individuals (①~⑨) and infertility inducing composition (①~⑦).
표 Table 1. Primer sets to identify candidate target sequence
표 Table 2. Primer sets to identify candidate target sequence (continuous)
표 Table 3. Forward primers to create the DNA template for sgRNA
표 Table 4. Raw data stats
표 Table 5. Trimming data stats
표 Table 6. Contig data stats
그림 Figure 2. Gene Ontology (GO) analysis. (A) Categorization of GO terms, (B) Biological process terms, (C) Cellular component terms, (D) Molecular function terms. All of the differentially expressed genes were classified based on GO analysis. Each category is labeled with different colors, and the numbers refer to ratio of these categories to the all dataset.
표 Table 7. 1^st selected Contig information
표 Table 8. 2^nd selected Contig information
그림 Figure 3. Candidate Target Sequence indicated in C69849_g1_i1 gene Sequence (1,357 bp). Yellow color for Candidate Target Sequence, Orange color for PAM (5'-NGG-3') region and Superscript for No. of Target sequence is Indicated.
그림 Figure 4. Candidate Target Sequence indicated in C82233_g1_i1 gene Sequence (531 bp). Yellow color for Candidate Target Sequence, Orange color for PAM (5'-NGG-3') region and Superscript for No. of Target sequence is Indicated.
그림 Figure 5. Candidate Target Sequence indicated in C183992_g1_i1 gene Sequence (324 bp). Yellow color for Candidate Target Sequence, Orange color for PAM (5'-NGG-3') region and Superscript for No. of Target sequence is Indicated.
그림 Figure 6-1. Candidate Target Sequence indicated in C63950_g1_i1 gene Sequence (2,850 bp). Yellow color for Candidate Target Sequence, Orange color for PAM (5'-NGG-3') region and Superscript for No. of Target sequence is Indicated.
그림 Figure 6-2. Candidate Target Sequence indicated in C63950_g1_i1 gene Sequence (2,850 bp). Yellow color for Candidate Target Sequence, Orange color for PAM (5'-NGG-3') region and Superscript for No. of Target sequence is Indicated.
그림 Figure 7. Gel electrophoresis of the sgRNA Templates (arrow: 130 bp band). (A) Lane1: 100 bp DNA marker, Lanes 2 to 9: sgRNA Templates for each target sequence No. 2, 3, 5, 6, 8, 9, 10, 11, 12, (B) Lane1: Marker, Lanes 2 to 12: sgRNA Templates for each target sequence No. 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, 25, 26.
표 Table 9. Target sequences for application of CRISPR/Cas9 system
표 Table 10. Synthesis of sgRNA
그림 Figure 8. sgRNA Screening Result by Target sequence of IZUMO1 (C69849_g1_i1) * Lane1: Marker, Lane2 : Uncleaved fragment, Lane3: B1 Cleaved fragments, Lane4: B2 Cleaved fragments. → Uf: Uncleaved fragment, → Cf: Cleaved fragments
그림 Figure 9. sgRNA Screening Result by Target sequence of IZUMO1 (C82233_g1_i1) * Lane1: 100bp DNA Marker, Lane2 : Uncleaved fragment, Lane3: B1 Cleaved fragments, Lane4: B2 Cleaved fragments. → Uf: Uncleaved fragment, → Cf: Cleaved fragments
그림 Figure 10. sgRNA Screening Result by Target sequence of IZUMO1 (C183992_g1_i1) * Lane1: 100bp DNA Marker, Lane2 : Uncleaved fragment, Lane3: B1 Cleaved fragments, Lane4: B2 Cleaved fragments. → Uf: Uncleaved fragment, → Cf: Cleaved fragments
그림 Figure 11-1. sgRNA Screening Result by Target sequence of Zona pellucida sperm-binding protein(C63950_g1_i1) * Lane1: 100bp DNA Marker, Lane2 : Uncleaved fragment, Lane3: B1 Cleaved fragments, Lane4: B2 Cleaved fragments. → Uf: Uncleaved fragment, → Cf: Cleaved fragments
그림 Figure 11-2. sgRNA Screening Result by Target sequence of Zona pellucida sperm-binding protein(C63950_g1_i1) * Lane1: 100bp DNA Marker, Lane2 : Uncleaved fragment, Lane3: B1 Cleaved fragments, Lane4: B2 Cleaved fragments. → Uf: Uncleaved fragment, → Cf: Cleaved fragments
그림 Figure 11-3. sgRNA Screening Result by Target sequence of Zona pellucida sperm-binding protein (C63950_g1_i1) * Lane1: 100bp DNA Marker, Lane2 : Uncleaved fragment, Lane3: B1 Cleaved fragments, Lane4: B2 Cleaved fragments. → Uf: Uncleaved fragment, → Cf: Cleaved fragments

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표제어: PCR

동의어: Packet Collision Rate

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용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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