화학공학소재연구정보센터
홈 로그인 로그아웃 연락처 사이트맵
Journal of Industrial and Engineering Chemistry, Vol.102, 241-250, October, 2021
DOI10.1016/j.jiec.2021.07.009  Export Citation
Cas9 conjugate complex delivering donor DNA for efficient gene editing by homology-directed repair
Yoo Kyung Kang1, Juhee Lee1, San Hae Im1, Joo Hoon Lee2, Juhee Jeong3, Duk Ki Kim3, 4, Seung Yun Yang5, Keehoon Jung3, 6, †, Sang-Gyu Kim1, †, and Hyun Jung Chung1, 2, †
  • 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea
  • 2Graduate School of Nanoscience and Technology, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea
  • 3Department of Anatomy and Cell Biology, Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea
  • 4Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea
  • 5Department of Biomaterials Science (BK21 Four Program), College of Natural Resources & Life Science, Pusan National University, Miryang, Republic of Korea
  • 6Institute of Allergy and Clinical Immunology, Seoul National University Medical Research Center, Seoul, Republic of Korea
E-mail:, ,
Delivery of the CRISPR ribonucleoprotein (RNP) for homology-directed repair (HDR) has been challenging due to the low efficiency. Herein, we developed a Cas9 conjugate complex system which can induce efficient HDR editing with the use of a minimal amount of carrier material. Cas9 from Streptococcus pyogenes was purified and conjugated with low molecular weight polymer (LP). The Cas9-LP conjugates were complexed with single guide RNA (sgRNA) and donor DNA, which showed greatly enhanced internalization into cells compared to native Cas9 complexes, as well as a high extent of co-localization of Cas9 with sgRNA. The cytotoxicity of Cas9-LP complexes was evaluated, demonstrating low cytotoxicity compared to the conventional lipofectamine formulation. Finally, the treatment of Cas9-LP complexes to HEK293T reporter cell line expressing a mutant red fluorescent protein (RFP) results in efficient base correction of the RFP gene (up to 31%), leading to restoration of RFP expression and fluorescence. We anticipate that the current method can be widely used as a platform for efficient HDR editing via ‘minimal carrierassisted’ delivery without the aid of any external physical stimuli, which can be potentially applied for in vivo and ex vivo editing of cellular targets.
Keywords:Cas9 conjugate;Ribonucleoprotein;Low molecular weight polymer;Homology-directed repair;Gene editing
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