Cas9 conjugate complex delivering donor DNA for efficient gene editing by homology-directed repair

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...

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Veröffentlicht in:Journal of industrial and engineering chemistry (Seoul, Korea) 2021, 102(0), , pp.241-250
Hauptverfasser: Kang, Yoo Kyung, Lee, Juhee, Im, San Hae, Lee, Joo Hoon, Jeong, Juhee, Kim, Duk Ki, Yang, Seung Yun, Jung, Keehoon, Kim, Sang-Gyu, Chung, Hyun Jung
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Sprache:eng
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Zusammenfassung: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 carrier-assisted’ delivery without the aid of any external physical stimuli, which can be potentially applied for in vivo and ex vivo editing of cellular targets.
ISSN:1226-086X
1876-794X
DOI:10.1016/j.jiec.2021.07.009