{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T19:24:38Z","timestamp":1779218678078,"version":"3.51.4"},"reference-count":54,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,10,18]],"date-time":"2021-10-18T00:00:00Z","timestamp":1634515200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["SP1600\/4-1 CRC1279 316249678-CRC1279 (project C02)"],"award-info":[{"award-number":["SP1600\/4-1 CRC1279 316249678-CRC1279 (project C02)"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002347","name":"Bundesministerium f\u00fcr Bildung und Forschung","doi-asserted-by":"publisher","award":["IMMUNOMOD"],"award-info":[{"award-number":["IMMUNOMOD"]}],"id":[{"id":"10.13039\/501100002347","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Pharmacol."],"abstract":"<jats:p>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 <jats:italic>Bacillus anthracis<\/jats:italic> 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.<\/jats:p>","DOI":"10.3389\/fphar.2021.770283","type":"journal-article","created":{"date-parts":[[2021,10,20]],"date-time":"2021-10-20T05:09:35Z","timestamp":1634706575000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":11,"title":["CRISPA: A Non-viral, Transient Cas9 Delivery System Based on Reengineered Anthrax Toxin"],"prefix":"10.3389","volume":"12","author":[{"given":"Maximilian","family":"Hirschenberger","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicole","family":"Stadler","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maximilian","family":"Fellermann","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Konstantin M. 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