*4.3. Detection of On-Target and O*ff*-Target Mutations*

The potential off-targets of sgRNAs were predicted using the "offTarget" program in the CRISPR-GE software toolkit [52]. Genomic DNA of rice was extracted using the CTAB (cetyl trimethylammonium bromide) method. The DNA fragments covering the on-target and off-target sites were amplified by PCR using the specific genomic primers. PCR amplifications were performed in a Mastercycler nexus gradient thermal cycler (Eppendorf, Hamburg, Germany). Each reaction contained DNA templates, 1 <sup>×</sup> PCR buffer, 0.4 mmol L−<sup>1</sup> dNTPs (deoxynucleotide triphosphates), 0.3 <sup>μ</sup>mol L−<sup>1</sup> of both forward and reverse primers, and 1 U KOD FX DNA polymerase (Toyobo, Osaka, Japan). Distilled water was added to a final volume of 50 μL. The PCR conditions included an initial incubation at 94 ◦C for 2 min, followed by 30 cycles of 98 ◦C for 10 s, 50–55 ◦C for 30 s, and 68 ◦C for 0.5–1 min, with a final extension at 68 ◦C for 5 min. The amplified products were sequenced directly. For some samples, PCR products were cloned and individual clones were sequenced. The superimposed sequencing chromatograms of heterozygous and bi-allelic mutations were decoded using DSDecodeM [53]. The PCR primers used are listed in Table S9.

## *4.4. Detection of the T-DNA Fragment*

The T-DNA fragment in transgenic plants was detected by PCR using three pairs of primers amplifying the *HPT*, sgRNA, and *Cas9* transgenes. Amplifications were carried out in a Mastercycler nexus gradient thermal cycler (Eppendorf, Hamburg, Germany). Each reaction contained DNA templates, 1 <sup>×</sup> Es Taq MasterMix (Cwbio, Beijing, China), and 0.4 <sup>μ</sup>mol L−<sup>1</sup> of both forward and reverse primers. Distilled water was added to a final volume of 25 μL. The PCR conditions included an initial incubation at 94 ◦C for 2 min, followed by 30 cycles of 94 ◦C for 30 s, 55 ◦C for 30 s, and 72 ◦C for 30 s, with a final extension at 72 ◦C for 2 min. PCR products were separated by electrophoresis on 1.5% agarose gels containing GoldView I nucleic acid dye (Solarbio, Beijing, China). The primers used are listed in Table S9.
