*2.4. Analysis of O*ff*-Target E*ff*ects*

To investigate the potential off-target events in our experiments, the two most probable off-target sites were selected for each of the 15 targets. The potential editing events in these sites were examined in all T0 plants and several randomly selected T1 lines. When the same off-target sequence occurred at different genomic loci, only one locus was examined. Of the 30 putative off-target sites, we detected mutations in 13 sites (Table 3). All the five loci that had a single-base mismatch with sgRNA exhibited off-target effects. Mutations were detected in four of the five (80.0%) and three of the 10 (30.0%) loci that had two and three mismatches, respectively. Additionally, one locus (Osfwl2aOFF-2) that had four dispersed mismatches was cleaved in transgenic plants. No mutations were detected in the loci that had five mismatches with sgRNA (Table 3). Most of the off-target sites with mutations were located within the gene region (Table 3).

*Int. J. Mol. Sci.* **2020**, *21*, 809


**Table 3.** Off-target effect analysis of transgenic plants. 1 The PAM sequences are shown in green and the mismatched bases in red.

Among the putative off-target sites with two mismatches, the mismatched nucleotides of Osfwl6bOFF-1 were separated by only one nucleotide in the PAM-proximal region, and no modifications were detected at this site (Table 3). Genotype analysis of the off-target mutations in T0 plants revealed that 44.9% and 43.5% of the genotypes were bi-alleles and heterozygotes, respectively (Figure 1C). Most off-target mutation events were insertions and deletions as observed in the on-target mutation events (Figure 1A,D).
