*2.3. Maize Protoplast Transformation*

Maize protoplasts were gene-edited by introducing CRISPR-Cas9 RNP complex (no integration of exogenous DNA) via PEG-mediated transfection (Figure 1). Protoplast transformation was adapted from Woo et al. [12] and Malnoy et al. [13]. First, 15 μg of the two components of the gRNA (crRNA and trans-activating crispr RNA-tracrRNA)) were incubated at 95 ◦C for 5 min. After allowing them to cool at room temperature, 45 μg of Cas9 and 1 × NEBuffer 3 were added, then mixed, and incubated at 25 ◦C for 10 min. Finally, the RNP complex was mixed with 100 μL of protoplasts (1 × 10<sup>5</sup> protoplasts), 250 μL of PEG solution (40% PEG 4000, 0.2 M mannitol, 0.1 M CaCl2) (pH 6.0), and incubated at 25 ◦C in the dark. Two incubation times were tested: T1 = 20 min and T2 = 40 min W5 solution (950 ul) was added and the tubes were centrifuged at 100 g for 3 min. Protoplasts were resuspended in 1 mL W1 solution [4 mM MES (pH 5.7), 0.5 M mannitol, 20 mM KCl], and then transferred to multi-well plates for 24 h under gentle agitation (40 rpm) in the dark at 25 ◦C.

**Figure 1.** Methodological approach of the present study. Maize seeds were germinated *in vitro*; the second leaves of the seedlings were used to obtain the protoplasts. The protoplasts were exposed to the CRISPR-Cas9 ribonucleoproteins complex (RNP), and after 24 h, the DNA was extracted from the samples. PCR fragments were amplified and sequenced. Maize image was taken from the free repository from pexel.com.
