Development and Research of Fanzor Genome Editing Tools in Dicot Crops

Firstly, we compared four Fanzors (SpuFz1, NlovFz2, MmeFz2, and MmFNuc) for genome engineering in tomato, which SpuFz1, NloFz2, and MmFNuc could perform targeted editing with editing efficiencies comparable to CRISPR-Cas9 in protoplasts. In tomato plants, SpuFz1, NlovFz2, and MmFNuc could produce edited plants, which the editing efficiency of NlovFz2 was 20.8%, and its editing efficiency at the SlSGR1 locus was superior to CRISPR-Cas9, indicating that Fanzor has good application potential for tomato. Subsequently, by introduced 1 or 2nt mismatches into the sgRNA guide sequence, we found that Fanzor is prone to show indel activity when the mismatch is located at the distal end of the TAM, indicating a lower specificity. However, no off-target editing was detected at potential off-target sites in the edited plants, suggesting the high genome-editing fidelity of Fanzor. Further, in peanut protoplasts, NlovFz2, MmeFz2, and MmFNuc could perform targeted editing. The different performances of SpuFz1 and MmeFz2 in tomato and peanut suggest that the editing capabilities of different Fanzor vary among species.