Axel O. Vera, Nicholas L. Truex, Vedagopuram Sreekanth, Bradley L. Pentelute, Amit Choudhary, and Ronald T. Raines 

PNAS; August 4, 2025; 122 (32) e2426960122; https://doi.org/10.1073/pnas.2426960122

Significance

The U.S. Food and Drug Administration recently approved the first CRISPR-Cas9 therapy, making genome editing a promising strategy for treating genetic disorders ex vivo and in vivo. Unintended or off-target genome editing increases with the cellular half-life of Cas9, is challenging to detect, and has unknown long-term consequences. We developed a cell-permeable anti-CRISPR protein system to “turn off” Cas9 after it achieves its intended genome editing outcome. Our technology reduces the half-life of Cas9 in human cells and its off-target activity and increases its genome-editing specificity.

Abstract

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, LF_N-Acr/PA, which delivers Acrs into cells within minutes. LF_N-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 (LF_N-Acr) binds and uses to enter the cell. In the presence of PA, LF_N-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 LF_N-Acr delivery reduces off-target base editing and increases Cas9 specificity by 41%. LF_N-Acr/PA is the most potent known cell-permeable CRISPR-Cas inhibition system, significantly improving the utility of CRISPR for genome editing.

See https://www.pnas.org/doi/10.1073/pnas.2426960122

Figure 1:

Mechanism of the anthrax protective antigen (PA)-mediated delivery of LF_N-Acr into cells. 1) Wild-type (WT) PA (i.e., PA 83 kDa: PA₈₃) binds to the ANTXR1 or ANTXR2 receptors on the cell surface. 2) Cell-surface proteases cleave and activate receptor-bound PA₈₃. 3) The activated PA (i.e., PA 63 kDa: PA₆₃) oligomerizes into a prepore. 4) The nontoxic N-terminal domain of lethal factor (LF_N) in the LF_N-Acr fusion binds to the PA prepore. 5) The entire complex is endocytosed. Acidification of the endosome promotes the PA prepore to change into a transmembrane pore, which translocates LF_N-Acr into the cytosol.