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

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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.

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Acknowledgements

This research was supported by the National Research Foundation (NRF) funded by Korean Government (Ministry of Science and ICT) (No. 2015R1C1A1A01052831 and 2017M3A9C6031798) and 2017 Research Grant from Kangwon National University (No. 520170405).

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  1. Yu Jin Kim and Hyelim Lee contributed equally to this study.

Authors and Affiliations

  1. Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon, 24341, Korea

    Yu Jin Kim, Hyelim Lee, Hyeonjin Cha & Ju Hyun Park

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  1. Yu Jin Kim
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Correspondence to Ju Hyun Park.

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Kim, Y.J., Lee, H., Cha, H. et al. Non-viral Gene Disruption by CRISPR/Cas9 Delivery Using Cell-permeable and Protein-stabilizing 30Kc19 Protein. Biotechnol Bioproc E 25, 724–733 (2020). https://doi.org/10.1007/s12257-020-0068-8

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