Membrane permeabilizing amphiphilic peptide delivers recombinant transcription factor and CRISPR-Cas9/Cpf1 ribonucleoproteins in hard-to-modify cells

PLoS One. 2018 Apr 4;13(4):e0195558. doi: 10.1371/journal.pone.0195558. eCollection 2018.

Abstract

Delivery of recombinant proteins to therapeutic cells is limited by a lack of efficient methods. This hinders the use of transcription factors or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleoproteins to develop cell therapies. Here, we report a soluble peptide designed for the direct delivery of proteins to mammalian cells including human stem cells, hard-to-modify primary natural killer (NK) cells, and cancer cell models. This peptide is composed of a 6x histidine-rich domain fused to the endosomolytic peptide CM18 and the cell penetrating peptide PTD4. A less than two-minute co-incubation of 6His-CM18-PTD4 peptide with spCas9 and/or asCpf1 CRISPR ribonucleoproteins achieves robust gene editing. The same procedure, co-incubating with the transcription factor HoxB4, achieves transcriptional regulation. The broad applicability and flexibility of this DNA- and chemical-free method across different cell types, particularly hard-to-transfect cells, opens the way for a direct use of proteins for biomedical research and cell therapy manufacturing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Cells, Cultured
  • Cerebral Cortex / cytology
  • Cerebral Cortex / metabolism
  • Cricetulus
  • Cytosol / metabolism
  • Endocytosis
  • Escherichia coli
  • Gene Editing / methods*
  • Humans
  • Mice
  • Myoblasts / cytology
  • Myoblasts / metabolism
  • Rats
  • Recombinant Proteins / administration & dosage
  • Recombinant Proteins / metabolism
  • Stem Cells / cytology
  • Stem Cells / metabolism
  • Transcription Factors / administration & dosage*
  • Transcription Factors / metabolism

Substances

  • Recombinant Proteins
  • Transcription Factors