ENAGenomicsInstitute of Biophysics, Chinese Academy of Scienceshttps://www.ebi.ac.uk/ena/browser/view/PRJNA1292198IscB, as the putative ancestors of Cas9, possesses a compact size, making it suitable for in vivo delivery. OgeuIscB is the first IscB protein to function in eukaryotic cells but requires a complicated TAM (NWRRNA). Here, we characterized a CRISPR-associated IscB system, named DelIscB, which recognizes a flexible TAM (NAC). Through systematically engineering its protein and sgRNA, we obtained enDelIscB with an average 48.9-fold increase in activity. By fusing enDelIscB with T5 exonuclease (T5E), we found enDelIscB-T5E displayed robust efficiency comparable to that of enIscB-T5E in human cells. Moreover, by fusing cytosine or adenosine deaminase with enDelIscB nickase, we established efficient miniature base editors (ICBE and IABE). Finally, we efficiently generated mouse models by microinjecting mRNA/sgRNA of enDelIscB and enDelIscB-T5E in mouse embryos. Collectively, our work presents a novel set of enDelIscB-based miniature tools with great potential for diverse applications in vivo.ENAfalsehumanEngineering a CRISPR-associated IscB system for developing miniature genome-editing tools2025-09-082025-09-08PRJNA1292198