Protective antigen-mediated delivery of an anti-CRISPR protein for precision genome editing

• 成果类型: Article
• 署名作者: Vera, Axel O.; Truex, Nicholas L.; Sreekanth, Vedagopuram; Pentelute, Bradley L.; Choudhary, Amit; Raines, Ronald T.
• 署名单位: Massachusetts Institute of Technology (MIT); Harvard University; Massachusetts Institute of Technology (MIT); Broad Institute; Harvard University; Harvard University Medical Affiliates; Brigham & Women's Hospital; Harvard University; Harvard University Medical Affiliates; Brigham & Women's Hospital; Harvard University; Harvard Medical School; Massachusetts Institute of Technology (MIT); University of South Carolina System; University of South Carolina Columbia
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-15233
• DOI: 10.1073/pnas.2426960122
• 发表日期: 2025-08-12
• 关键词: anthrax toxin; mammalian-cells; dna; inhibition; receptor; CHALLENGES; mechanism; cleavage; base; ACID

摘要

Precise control over the dosage of Cas9-based technologies is essential because off-target effects, mosaicism, chromosomal aberrations, immunogenicity, and genotoxicity can arise with prolonged Cas9 activity. Type II anti-CRISPR proteins (Acrs) inhibit and control Cas9 but are generally impermeable to the cell membrane due to their size and anionic charge. Moreover, existing Acr delivery methods are long-lived and operate within hours (e.g., viral and nonviral vectors) or require external devices (e.g., electroporation), limiting therapeutic applications. To address these problems, we developed a protein-based anti-CRISPR delivery platform, LFN-Acr/PA, which delivers Acrs into cells within minutes. LFN-Acr/PA is a nontoxic, two-component protein system derived from anthrax toxin, where protective antigen (PA) proteins bind receptors widespread in human cells, forming a pH-triggered endosomal pore that an engineered Acr (LFN-Acr) binds and uses to enter the cell. In the presence of PA, LFN-Acr enters human cells (e.g., immortalized cell lines, embryonic stem cells, and 3D cell cultures) at concentrations as low as 2.5 pM to inhibit up to 95% of Cas9-mediated knockout, knock-in, transcriptional activation, and base editing. Timing LFN-Acr delivery reduces off-target base editing and increases Cas9 specificity by 41%. LFN-Acr/PA is the most potent known cell-permeable CRISPR-Cas inhibition system, significantly improving the utility of CRISPR for genome editing.