Ingeniería de nucleasas IscB hipercompactas para la edición genómica eficiente y versátil en arroz

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.

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.

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.