Molecular Therapy: Nucleic Acids (Jun 2022)
Highly specific chimeric DNA-RNA-guided genome editing with enhanced CRISPR-Cas12a system
- Hanseop Kim,
- Wi-jae Lee,
- Chan Hyoung Kim,
- Yeounsun Oh,
- Lee Wha Gwon,
- Hyomin Lee,
- Woojeung Song,
- Junho K. Hur,
- Kyung-Seob Lim,
- Kang Jin Jeong,
- Ki-Hoan Nam,
- Young-Suk Won,
- Kyeong-Ryoon Lee,
- Youngjeon Lee,
- Young-Hyun Kim,
- Jae-Won Huh,
- Bong-Hyun Jun,
- Dong-Seok Lee,
- Seung Hwan Lee
Affiliations
- Hanseop Kim
- Futuristic Animal Resource & Research Center (FARRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Republic of Korea; School of Life Sciences and Biotechnology, BK21 FOUR KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea
- Wi-jae Lee
- Futuristic Animal Resource & Research Center (FARRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Republic of Korea; Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea
- Chan Hyoung Kim
- Futuristic Animal Resource & Research Center (FARRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Republic of Korea; Department of Biological Sciences, Chungnam National University, Daejeon, South Korea
- Yeounsun Oh
- Futuristic Animal Resource & Research Center (FARRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Republic of Korea; Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea
- Lee Wha Gwon
- National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Republic of Korea; Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology, Gajeong-dong, Yuseong-gu, Daejeon, Republic of Korea
- Hyomin Lee
- Department of Medicine, Major in Medical Genetics, Graduate School, Hanyang University, Seoul 04763, Republic of Korea
- Woojeung Song
- Department of Medicine, Major in Medical Genetics, Graduate School, Hanyang University, Seoul 04763, Republic of Korea
- Junho K. Hur
- Department of Genetics, College of Medicine, Hanyang University, Seoul 04763, Republic of Korea; Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, Republic of Korea
- Kyung-Seob Lim
- Futuristic Animal Resource & Research Center (FARRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Republic of Korea
- Kang Jin Jeong
- National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Republic of Korea
- Ki-Hoan Nam
- Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Republic of Korea
- Young-Suk Won
- Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Republic of Korea
- Kyeong-Ryoon Lee
- Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Republic of Korea; Department of Bioscience, University of Science and Technology, Daejeon 34113, Republic of Korea
- Youngjeon Lee
- National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Republic of Korea
- Young-Hyun Kim
- National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Republic of Korea
- Jae-Won Huh
- National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Republic of Korea; Department of Functional Genomics, KRIBB School of Bioscience, Korea University of Science & Technology (UST), Daejeon, Republic of Korea
- Bong-Hyun Jun
- Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea; Corresponding author Bong-Hyun Jun, PhD, Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Korea.
- Dong-Seok Lee
- School of Life Sciences and Biotechnology, BK21 FOUR KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea; Corresponding author Dong-Seok Lee, PhD, School of Life Sciences and Biotechnology, BK21 FOUR KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea.
- Seung Hwan Lee
- National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Republic of Korea; Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology, Gajeong-dong, Yuseong-gu, Daejeon, Republic of Korea; Department of Life Science, Chung-Ang University, Seoul 06974, Republic of Korea; Corresponding author Seung Hwan Lee, PhD, Department of Life Science, Chung-Ang University, Seoul 06974, Republic of Korea.
- Journal volume & issue
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Vol. 28
pp. 353 – 362
Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system is composed of a Cas12a effector that acts as a DNA-cleaving endonuclease and a crispr RNA (crRNA) that guides the effector to the target DNA. It is considered a key molecule for inducing target-specific gene editing in various living systems. Here, we improved the efficiency and specificity of the CRISPR-Cas12a system through protein and crRNA engineering. In particular, to optimize the CRISPR-Cas12a system at the molecular level, we used a chimeric DNA-RNA guide chemically similar to crRNA to maximize target sequence specificity. Compared with the wild-type (wt)-Cas12a system, when using enhanced Cas12a system (en-Cas12a), the efficiency and target specificity improved on average by 2.58 and 2.77 times, respectively. In our study, when the chimeric DNA-RNA-guided en-Cas12a effector was used, the gene-editing efficiency and accuracy were simultaneously increased. These findings could contribute to highly accurate genome editing, such as human gene therapy, in the near future.
