• Journal of Plant Biotechnology
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• 제44권1호
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• pp.89-96
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• 2017

CRISPR/Cas9 기술은 생명공학을 활용한 신품종 작물육성에 있어 패러다임 변혁을 가져다 줄 핵심 기반기술이다. 본 연구에서는 CRISPR/Cas9를 이용하여 유전자편집기술을 기존에 알려진 방법보다 쉽고 정확하게 실험 할 수 있도록 sgRNA 디자인, 벡터구축, 형질전환체 육성 및 분석 등을 자세히 기술하였다. sgRNA는 http://www.rgenome.net/ 사이트에서 NGG 영역을 중심으로 하여 target-up: 5'-ggcaGNNNNNNNNNNNNNNNNNNNN-3'과 target-down: 5'-aaacNNNNNNNNNNNNNNNNNNNNC-3'의 올리고를 디자인하였다. 식물형질전환용 벡터는 pPZP-Cas9-RGEN을 기본으로 하였으며, sgRNA의 프로모터는 OsU3를 이용하여 pPZP::35S::Cas9::PinII-OsU3::sgRNA::Bar-Gen 순으로 구축하였다. 형질전환체의 육성은 단기형질전환 Agrobacterium 법을 사용하였으며 재분화 식물체를 얻는데48일 정도 소요되었다. 형질전환체 유무는 genomic PCR 분석으로 single copy 선발은 TaqMan PCR로 분석하였다. 정밀유전자편집 식물체는 T1 세대에서 T-DNA 삽입되지 않은 식물체를 Bar-strip에 의해 선발하였다. 선발된 식물체의 sgRNA 영역의 염기배열 조사에 의해 유전자 편집 식물체를 육성하였다. 따라서 본 연구에서 CRISPR/Cas9 system에 의한 정밀유전자편집 기술을 이용하여 보다 빠르고 쉽고 경제적으로 유전자가 편집된 개체를 확보할 수 있었다. 본 실험에서 확립된 system은 상업용 식물 계통육성에 이용 가능하여 육종적 가치가 매우 클 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제34권7호
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• pp.1522-1529
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• 2024

CRISPR-Cas system is being used as a powerful genome editing tool with developments focused on enhancing its efficiency and accuracy. Recently, the miniature CRISPR-Cas12f1 system, which is small enough to be easily loaded onto various vectors for cellular delivery, has gained attention. In this study, we explored the influence of temperature conditions on multiplex genome editing using CRISPR-Cas12f1 in an Escherichia coli model. It was revealed that when two distinct targets in the genome are edited simultaneously, the editing efficiency can be enhanced by allowing cells to recover at a reduced temperature during the editing process. Additionally, employing 3'-end truncated sgRNAs facilitated the simultaneous single-nucleotide level editing of three targets. Our results underscore the potential of optimizing recovery temperature and sgRNA design protocols in developing more effective and precise strategies for multiplex genome editing across various organisms.

• Journal of Microbiology and Biotechnology
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• 제30권12호
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• pp.1919-1926
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• 2020

CRISPR interference (CRISPRi) has been developed as a transcriptional control tool by inactivating the DNA cleavage ability of Cas9 nucleases to produce dCas9 (deactivated Cas9), and leaving dCas9 the ability to specifically bind to the target DNA sequence. CRISPR/Cas9 technology has limitations in designing target-specific single-guide RNA (sgRNA) due to the dependence of protospacer adjacent motif (PAM) (5'-NGG) for binding target DNAs. Reportedly, Cas9-NG recognizing 5'-NG as the PAM sequence has been constructed by removing the dependence on the last base G of PAM through protein engineering of Cas9. In this study, a dCas9-NG protein was engineered by introducing two active site mutations in Cas9-NG, and its ability to regulate transcription was evaluated in the gal promoter in E. coli. Analysis of cell growth rate, D-galactose consumption rate, and gal transcripts confirmed that dCas9-NG can completely repress the promoter by recognizing DNA targets with PAM of 5'-NGG, NGA, NGC, NGT, and NAG. Our study showed possible PAM sequences for dCas9-NG and provided information on target-specific sgRNA design for regulation of both gene expression and cellular metabolism.