Engineering a CRISPR-associated IscB system for developing miniature genome-editing tools in human cells and mouse embryos
Year: 2025
Type: article
Abstract: IscB, as the putative ancestor of Cas9, possesses a compact size, making it suitable for in vivo delivery. OgeuIscB is the first IscB protein known to function in eukaryotic cells but requires a complex TAM (NWRRNA). Here, we characterize a CRISPR-associated IscB system, named DelIscB, which recognizes a flexible TAM (NAC). Through systematically engineering its protein and sgRNA, we obtain enDelIscB with an average 48.9-fold increase in activity. By fusing enDelIscB with T5 exonuclease (T5E), we find that enDelIscB-T5E displays robust efficiency comparable to that of enIscB-T5E in human cells. Moreover, by fusing cytosine or adenosine deaminase with enDelIscB nickase, we establish efficient miniature base editors (ICBE and IABE). Finally, we efficiently generate mouse models by microinjecting mRNA/sgRNA of enDelIscB and enDelIscB-T5E into mouse embryos. Collectively, our work presents a set of enDelIscB-based miniature genome-editing tools with great potential for diverse applications in vivo. Cas9-based genome editing tools face challenges for efficient in vivo delivery due to their large size. Here, the authors present a set of compact genome-editing tools engineered from a CRISPR-associated IscB system which exhibit robust editing efficiency in human cells and mouse embryos.
Source: Nature Communications
Authors: Fei Zhang, Yuxin Peng, Dongdong Fan, Guoxu Song, Xing Gao, Yong Tian
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $6,990
OpenAlex ID: W4416756770