*2.4. Gene-Editing E*ffi*ciency Analysis by Sanger Sequencing*

In order to characterize the spectrum and frequency of DNA changes at the target gene, genomic DNA was isolated using DNeasy ® Plant Mini Kit (QIAGEN ® Biotecnologia Brasil Ltd., Sao Paulo, SP, Brazil), followed by amplification of the target region by PCR using Taq Q5 High-Fidelity DNA Polymerase (NEB ®, Ipswich, MA, USA) and primers listed in Table 1. PCR samples were purified and sequenced using the BigDye Terminator 3.1v Kit (ThermoFisher Scientific, Sao Paulo, SP, Brazil). Samples were resuspended in formamide, denatured at 95 ◦C for 5 min, and incubated on ice for 3 min. Sequencing was performed using the Sanger [22] automated sequencer from the 3500xL Dx Genetic Analyzer for Sequencing (Applied BiosystemsTM-ThermoFisher Scientific, Sao Paulo, SP, Brazil).

CRISPR-Cas9 DNA changes were calculated based on the insertions and deletions (In/Dels) around the cleavage site (3 bp upstream of the PAM sequence) using the Inference of CRISPR Editing Software—ICE software v.2 (Synthego Corporation, Palo Alto, CA, USA). It has been previously shown that the ICE software results are comparable to the Next Generation Sequencing (NGS) results [23].
