Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA.
Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA.
Nat Biotechnol. 2024 Feb;42(2):305-315. doi: 10.1038/s41587-023-01756-1. Epub 2023 Apr 24.
Simple, efficient and well-tolerated delivery of CRISPR genome editing systems into primary cells remains a major challenge. Here we describe an engineered Peptide-Assisted Genome Editing (PAGE) CRISPR-Cas system for rapid and robust editing of primary cells with minimal toxicity. The PAGE system requires only a 30-min incubation with a cell-penetrating Cas9 or Cas12a and a cell-penetrating endosomal escape peptide to achieve robust single and multiplex genome editing. Unlike electroporation-based methods, PAGE gene editing has low cellular toxicity and shows no significant transcriptional perturbation. We demonstrate rapid and efficient editing of primary cells, including human and mouse T cells, as well as human hematopoietic progenitor cells, with editing efficiencies upwards of 98%. PAGE provides a broadly generalizable platform for next-generation genome engineering in primary cells.
将 CRISPR 基因组编辑系统简单、高效且良好地递送至原代细胞仍然是一个主要挑战。在这里，我们描述了一种经过工程改造的肽辅助基因组编辑 (PAGE) CRISPR-Cas 系统，用于快速、稳健地编辑原代细胞，毒性最小。PAGE 系统仅需 30 分钟与细胞穿透性 Cas9 或 Cas12a 以及细胞穿透性内涵体逃逸肽孵育，即可实现稳健的单基因和多基因编辑。与基于电穿孔的方法不同，PAGE 基因编辑具有低细胞毒性，并且不会引起明显的转录扰动。我们证明了 PAGE 对原代细胞（包括人类和小鼠 T 细胞以及人类造血祖细胞）的快速高效编辑，编辑效率高达 98%以上。PAGE 为原代细胞的下一代基因组工程提供了一个广泛适用的平台。
