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.

由于效率低，用于同源定向修复 (HDR) 的 CRISPR 核糖核蛋白 (RNP) 的递送一直具有挑战性。在此，我们开发了一种 Cas9 共轭复合系统，该系统可以使用最少量的载体材料来诱导高效的 HDR 编辑。来自化脓性链球菌的Cas9纯化并与低分子量聚合物 (LP) 结合。Cas9-LP 偶联物与单向导 RNA (sgRNA) 和供体 DNA 复合，与天然 Cas9 复合物相比，其显示出大大增强的细胞内化，以及 Cas9 与 sgRNA 的高度共定位。对 Cas9-LP 复合物的细胞毒性进行了评估，证明与传统的 lipofectamine 制剂相比，细胞毒性较低。最后，将 Cas9-LP 复合物与表达突变红色荧光蛋白 (RFP) 的 HEK293T 报告细胞系进行处理，导致 RFP 基因的有效碱基校正（高达 31%），导致 RFP 表达和荧光的恢复。细胞靶标的体内和体外编辑。