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Non-viral Gene Disruption by CRISPR/Cas9 Delivery Using Cell-permeable and Protein-stabilizing 30Kc19 Protein

Biotechnology and Bioprocess Engineering 2020년 25권 5호 p.724 ~ 733
김유진, 이혜림, 차현진, 박주현,
소속 상세정보
김유진 ( Kim Yu-Jin ) - Kangwon National University Department of Medical Biomaterials Engineering
이혜림 ( Lee Hye-Lim ) - Kangwon National University Department of Medical Biomaterials Engineering
차현진 ( Cha Hyeon-Jin ) - Kangwon National University Department of Medical Biomaterials Engineering
박주현 ( Park Ju-Hyun ) - Kangwon National University Department of Medical Biomaterials Engineering

Abstract


CRISPR/Cas9 system has served a new insight in genome editing of eukaryotes, including human cells. In this system, delivery of Cas9 nuclease with guide RNA has been central challenge in developing safe and efficient techniques. The viral delivery of genes encoding these two components i.e. Cas9 and guide RNA may cause unexpected integration of the DNA sequence into the host cell genome, and lead to potential safety problems such as tumorigenesis. Herein, we report that the Cas9 protein can be directly delivered into the human cells through fusion with 30Kc19, a cell-penetrating and protein-stabilizing protein originating from silkworm. The 30Kc19-conjugated Cas9 (30Kc19-Cas9) showed higher stability than native Cas9 for thermal and chemical-induced inactivation in DNA-cleavable activity. In addition, it was demonstrated that 30Kc19-Cas9 was efficiently delivered into human cells and resulted in targeted gene disruption with single guide RNA by showing gene expression and site-specific mutations in the genome. With the advantages of efficient delivery in addition to the enhancement of Cas9 stability, this method is expected to provide a versatile strategy to advance non-viral and clinically-feasible genome editing for in vivo applications.

키워드

CRISPR/Cas9; genome editing; protein delivery; protein stability; 30Kc19

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