Yucai Li, Chenfei Li, Jiaying Yang, Xinrong Sun, Jingying Li, Lei Yan, Chen Zhang, Shaoya Li, Yubing He, Lanqin Xia

Plant Communications; 2025; IF: 11.6

DOI:10.1016/j.xplc.2025.101562

Abstract

As the ancestors of Cas9 endonuclease from IS200/605 transposon, IscB has been received extensive attentions recently due to its smaller protein size of ~490 aa. However, whether the IscB and its enhanced versions (enIscBs) enabling efficient and heritable plant genome editing remains to be explored. In this study, five hypercompact miniature enIscB-enωRNA systems were engineered for rice genome editing by fusing T5 exonuclease (T5E) to the respective enIscBs and coupling with their associated optimized ωRNAs (enωRNAs), respectively. Systemic evaluations of these enIscB-enωRNA variants in rice protoplasts indicated that fusion of T5E to the respective enIscBs significantly increased their overall average editing efficiencies at the tested endogenous target sites, respectively. Among which, enIscBv1-T5E-enωRNAv1, enIscBv3-T5E-enωRNAv3, and enIscBv3-T5E-enωRNAv4 outperformed other enIscB-T5E-enωRNA variants both in rice protoplast and stable lines, albeit all of which exhibiting a severe target dependent manner. The editing efficiency could reach up to 41.87% in rice stable lines. Furthermore, fusion of T5E not only enlarged the editing window, but also induced indels and larger deletions. The engineered hypercompact miniature enIscB-enωRNA systems enrich the plant genome editing toolkits and thus have potential applications in crop improvement.