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.

CRISPR / Cas9系统为真核生物包括人类细胞的基因组编辑提供了新的见识。在该系统中，Cas9核酸酶与指导RNA的传递一直是开发安全有效技术的主要挑战。病毒传递编码这两个成分的基因，即Cas9和引导RNA可能会导致DNA序列意外整合到宿主细胞基因组中，并导致潜在的安全性问题，例如肿瘤发生。在此，我们报道Cas9蛋白可以通过与30Kc19融合而直接递送到人类细胞中，而30Kc19是一种来自蚕的细胞穿透性蛋白稳定蛋白。30Kc19偶联的Cas9（30Kc19-Cas9）对于DNA裂解活性的热和化学诱导失活显示出比天然Cas9高的稳定性。此外，通过显示基因组中的基因表达和位点特异性突变，证明了30Kc19-Cas9被有效地递送到人细胞中并导致了单个指导RNA的靶向基因破坏。除了增强Cas9的稳定性外，还具有高效交付的优势，体内应用。