Frontiers in Pharmacology (Oct 2021)

CRISPA: A Non-viral, Transient Cas9 Delivery System Based on Reengineered Anthrax Toxin

  • Maximilian Hirschenberger,
  • Maximilian Hirschenberger,
  • Nicole Stadler,
  • Maximilian Fellermann,
  • Konstantin M. J. Sparrer,
  • Frank Kirchhoff,
  • Holger Barth,
  • Panagiotis Papatheodorou

DOI
https://doi.org/10.3389/fphar.2021.770283
Journal volume & issue
Vol. 12

Abstract

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Translating the CRISPR/Cas9 genome editing technology into clinics is still hampered by rather unspecific, unsafe and/or inconvenient approaches for the delivery of its main components - the Cas9 endonuclease and a guide RNA - into cells. Here, we describe the development of a novel transient and non-viral Cas9 delivery strategy based on the translocation machinery of the Bacillus anthracis anthrax toxin, PA (protective antigen). We show that Cas9 variants fused to the N-terminus of the lethal factor or to a hexahistidine tag are shuttled through channels formed by PA into the cytosol of human cells. As proof-of-principle, we applied our new approach, denoted as CRISPA, to knock out lipolysis-stimulated lipoprotein receptor (LSR) in the human colon cancer cell line HCT116 and green-fluorescent protein (GFP) in human embryonic kidney 293T cells stably expressing GFP. Notably, we confirmed that the transporter PA can be adapted to recognize specific host cell-surface receptor proteins and may be optimized for cell type-selective delivery of Cas9. Altogether, CRISPA provides a novel, transient and non-viral way to deliver Cas9 into specific cells. Thus, this system is an additional step towards safe translation of the CRISPR/Cas9 technology into clinics.

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