Engineering hypercompact IscB nucleases for efficient and versatile genome editing in rice

BACKGROUND: IscB (Insertion sequences Cas9-like OrfB) represents a novel class of RNA-guided nucleases, approximately one-third the size of Cas9 proteins. Despite the limited natural efficiency in eukaryotic cells, recent advances have led to the engineering of several IscBs for mammalian genome editing. RESULTS: In this study, we screen and identify high-activity IscB variants for rice. A version of pIscB-v3, combining enOgeuIscB and ωRNA-v13, demonstrated superior mutagenesis efficiency compared to other systems. The average editing efficiency of pIscB-v3 is 17.61% from ten endogenous targets, and we obtain edited lines in up to 83.33% of T0 generation with 33.33% of homozygous and bi-allelic mutations. Further analysis reveals that pIscB-v3 exhibits high editing specificity and relaxed target-adjacent motif (TAM) compatibility in rice. Beyond gene knockout systems, we develop cytosine base editors (CBEs) and adenine base editors (ABEs) from pIscB-v3. We find that the ssDNA-targeting SCP1.201 family deaminase Sdd7 outperformed human APOBEC3A in IscB-CBEs for C-to-T conversions in rice. The Sdd7-nIscB achieves precise edits in 22.92% of lines on average, with a maximum frequency of 47.92%. Additionally, TadA8e-nIscB exhibits limited activity. However, fusing an extra copy of TadA-8e to either terminus of TadA8e-nIsc significantly enhances A-to-G conversions. CONCLUSIONS: Collectively, our results demonstrate the robust capabilities of IscB to develop an efficient and versatile miniature plant genome editing toolkit to substantially facilitate crop breeding.