Luo Xianjin, Weidinger Eric, Burghardt Tobias, Höhn Miriam, Wagner Ernst
Pharmaceutical Biotechnology, Department of Pharmacy, Ludwig-Maximilians-Universität Munich, Butenandtstrasse 5-13, 81377 Munich, Germany.
Center for Nanoscience (CeNS), LMU Munich, 80799 Munich, Germany.
Int J Mol Sci. 2025 May 3;26(9):4361. doi: 10.3390/ijms26094361.
CRISPR-Cas9 genome editing is a versatile platform for studying and treating various diseases. Homology-directed repair (HDR) with DNA donor templates serves as the primary pathway for gene correction in therapeutic applications, but its efficiency remains a significant challenge. This study investigates strategies to enhance gene correction efficiency using a T-shaped lipo-xenopeptide (XP)-based Cas9 RNP/ssDNA delivery system combined with various HDR enhancers. Nu7441, a known DNA-PKcs inhibitor, was found to be most effective in enhancing HDR-mediated gene correction. An over 10-fold increase in HDR efficiency was achieved by Nu7441 in HeLa-eGFPd2 cells, with a peak HDR efficiency of 53% at a 5 nM RNP concentration and up to 61% efficiency confirmed by Sanger sequencing. Surprisingly, the total gene editing efficiency including non-homologous end joining (NHEJ) was also improved. For example, Nu7441 boosted exon skipping via NHEJ-mediated splice site destruction by 30-fold in a DMD reporter cell model. Nu7441 modulated the cell cycle by reducing cells in the G1 phase and extending the S and G2/M phases without compromising cellular uptake or endosomal escape. The enhancement in genome editing by Nu7441 was widely applicable across several cell lines, several Cas9 RNP/ssDNA carriers (LAF-XPs), and also Cas9 mRNA/sgRNA/ssDNA polyplexes. These findings highlight a novel and counterintuitive role for Nu7441 as an enhancer of both HDR and total gene editing efficiency, presenting a promising strategy for Cas9 RNP-based gene therapy.
CRISPR-Cas9基因组编辑是用于研究和治疗各种疾病的通用平台。使用DNA供体模板的同源定向修复（HDR）是治疗应用中基因校正的主要途径，但其效率仍然是一项重大挑战。本研究调查了使用基于T形脂肽（XP）的Cas9核糖核蛋白/单链DNA递送系统与各种HDR增强剂相结合来提高基因校正效率的策略。发现已知的DNA-PKcs抑制剂Nu7441在增强HDR介导的基因校正方面最有效。在HeLa-eGFPd2细胞中，Nu7441使HDR效率提高了10倍以上，在5 nM核糖核蛋白浓度下，HDR效率峰值为53%，经桑格测序确认效率高达61%。令人惊讶的是，包括非同源末端连接（NHEJ）在内的总基因编辑效率也得到了提高。例如，在杜氏肌营养不良症（DMD）报告细胞模型中，Nu7441通过NHEJ介导的剪接位点破坏使外显子跳跃提高了30倍。Nu7441通过减少G1期细胞并延长S期和G2/M期来调节细胞周期，而不会影响细胞摄取或内体逃逸。Nu7441对基因组编辑的增强作用广泛适用于多种细胞系、多种Cas9核糖核蛋白/单链DNA载体（LAF-XP）以及Cas9信使核糖核酸/单向导RNA/单链DNA多聚体。这些发现突出了Nu7441作为HDR和总基因编辑效率增强剂的新的、与直觉相反的作用，为基于Cas9核糖核蛋白的基因治疗提供了一种有前景的策略。
