Kim, Seung Mm Shin, Sang Chul Kim, Eunice EunKyeong Kim, Sang-Heon Park, Kwideok Oh, Seung Ja Jang, Mihue 2024-01-19T22:03:38Z 2024-01-19T22:03:38Z 2021-09-03 2018-08 1936-0851 https://pubs.kist.re.kr/handle/201004/121081 Cas9 ribonucleoprotein (RNP)-mediated delivery has emerged as an ideal approach for in vivo applications. However, the delivery of Cas9 RNPs requires electroporation or lipid- or cationic-reagent-mediated transfection. Here, we developed a carrier-free Cas9 RNP delivery system for robust gene editing in vivo. For simultaneous delivery of Cas9 and a guide RNA into target cells without the aid of any transfection reagents, we established a multifunctional Cas9 fusion protein (Cas9-LMWP) that forms a ternary complex with synthetic crRNA:tracrRNA hybrids in a simple procedure. Cas9-LMWP carrying both a nuclear localization sequence and a low-molecular weight protamine (LMWP) enables the direct self-assembly of a Cas9:crRNA:tracrRNA ternary complex (a ternary Cas9 RNP) and allows for the delivery of the ternary Cas9 RNPs into the recipient cells, owing to its intrinsic cellular and nuclear translocation ability with low immunogenicity. To demonstrate the potential of this system, we showed extensive synergistic anti-KRAS therapy (CI value: 0.34) via in vitro and in vivo editing of the KRAS gene by the direct delivery of multifunctional Cas9 RNPs in lung cancer. Thus, our carrier-free Cas9 RNP delivery system could be an innovative platform that might serve as an alternative to conventional transfection reagents for simple gene editing and high-throughput genetic screening. English AMER CHEMICAL SOC DELIVERY RNA PROTEIN DNA PROTAMINE INHIBITORS MEMBRANE PATHWAYS PEPTIDE BRAIN Simple in Vivo Gene Editing via Direct Self-Assembly of Cas9 Ribonucleoprotein Complexes for Cancer Treatment Article 10.1021/acsnano.8b01670 1 ACS NANO, v.12, no.8, pp.7750 - 7760 ACS NANO 12 8 7750 7760 scie scopus 000443525600029 2-s2.0-85050637271 Chemistry, Multidisciplinary Chemistry, Physical Nanoscience & Nanotechnology Materials Science, Multidisciplinary Chemistry Science & Technology - Other Topics Materials Science Article DELIVERY RNA PROTEIN DNA PROTAMINE INHIBITORS MEMBRANE PATHWAYS PEPTIDE BRAIN CRISPR/Cas9 genome editing low-molecular-weight protamine (LMWP) cancer therapy in vivo gene editing