한빛사 논문
Hanseop Kim1,2,†, Wi-jae Lee1,3,†, Yeounsun Oh1,4, Seung-Hun Kang1,5, Junho K. Hur6,7,8,
Hyomin Lee7, WooJeung Song7, Kyung-Seob Lim1, Young-Ho Park1, Bong-Seok Song1,
Yeung Bae Jin9, Bong-Hyun Jun3, Cheulhee Jung4, Dong-Seok Lee2,*, Sun-Uk Kim1,10,* and Seung Hwan Lee9,*
1Futuristic Animal Resource & Research Center (FARRC), Korea Research Institute of Bioscience and Biotechnology(KRIBB), Cheongju, Korea
2School of Life Sciences and Biotechnology, BK21 Plus KNU Creative BioResearch
Group, Kyungpook National University, Daegu, Republic of Korea
3Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea
4Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea
5Department of Medicine, Graduate School, Kyung Hee University, Seoul 02447, Republic of Korea
6Department of Pathology, College of Medicine, Kyung Hee University, Seoul 02447, Republic of Korea
7Department of Biomedical Science, Graduate School, Kyung Hee University, Seoul 02447, Republic of Korea
8Department of Medical Genetics, College of Medicine, Hanyang University, Seoul, Republic of Korea
9National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Republic of Korea
10Department of Functional Genomics, KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon, Republic of Korea
†The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
*Corresponding author
Abstract
The CRISPR–Cas9 system is widely used for target-specific genome engineering. CRISPR–Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR–Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR–Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.
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