Abstract Targeted delivery of therapeutics to specific cells is a major bottleneck towards personalized medicine. The extracellular injection system (eCIS) of Serratia entomophila , the antifeeding prophage (Afp), promises potential for drug delivery purposes. However, the precise mechanism of action, toxin location, and Afp loading remain unclear. Here, we reveal a minimal N-terminal signal peptide (NtSP) of the toxin Afp18, that plays a key role in toxin packing. By engineering fusion proteins, we demonstrate that Afp18’s NtSP can shuttle effectors for Afp loading. We packed non-eCIS effectors, including CRISPR-Cas protein CasΦ-2 from Biggiephage, and a human antimicrobial peptide, LL37, into Afp. Additionally, NtSPs from eCIS effectors of other species facilitate loading of CasΦ-2 into Afp. We observed cargo being packed inside the Afp tail tube through cryo-EM single particle analysis. The presented results enhance our understanding of eCIS toxin packing and contribute to their development as targeted delivery systems. Teaser A novel use of the Afp nano injection system’s N-terminal signal peptide in targeted therapeutics delivery

N-terminal toxin signal peptides efficiently load therapeutics into a natural nano-injection system

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