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

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 (6–54 kDa) and anionic charge. Moreover, existing Acr delivery methods are long-lived and operate within hours ( e.g ., viral and non-viral vectors) or are not applicable in vivo ( e.g ., nucleofection), limiting therapeutic applications. To address these problems, we developed the first 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 proteins bind receptors widespread in human cells, forming a pH-triggered endosomal pore that LF _N -Acr binds and uses to enter the cell. In the presence of PA, LF _N -Acr enters human cells 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.