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.


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


  • Recombinant Proteins
  • Transcription Factors

Grants and funding

This work was supported in part by CNRC.NRC, Programme d'aide à la recherche industrielle, PARICNRC 874333 and by Feldan Therapeutics Inc. The funders provided support in the form of salaries for authors T.D., J.-P.L.-S., L.-J.B., J.R., C.L., V.T., X.B., D.G, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are related in the ‘author contributions’ section.