Efficient engineering of human and mouse primary cells using peptide-assisted genome editing

Nat Biotechnol. 2024 Feb;42(2):305-315. doi: 10.1038/s41587-023-01756-1. Epub 2023 Apr 24.

Abstract

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.

MeSH terms

  • Animals
  • CRISPR-Cas Systems* / genetics
  • Electroporation
  • Gene Editing* / methods
  • Hematopoietic Stem Cells
  • Humans
  • Mice