**2. Materials and Methods**

### *2.1. Target Site Selection and In Vitro Cleavage Assay*

The *Zea mays* IPK gene sequence data was obtained from the NCBI GenBank (accession B73RefGen\_v3). In vitro tests were performed to confirm the RNP complex e fficiency to cleave target DNA. The target site was amplified using specific primers. The crispr-RNAs (crRNAs) were designed for the third exon of the IPK gene in maize using the platform CRISPR-Cas9 guide RNA Design Checker (Integrated DNA Technologies Inc. IDT, Coralville, IA, SUA). Commercially available Cas9 protein (160 kDa) was also purchased from IDT (Table 1).


**Table 1.** List of primers and crispr-RNAs (crRNAs) used for the amplification and mutation of the *inositol phosphate kinase* gene (IPK) target locus in maize via CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9.

> Note: \* Selected crRNAs used for protoplasts transfection.

gRNA, crRNA (100 nM), and trans-activating crispr-RNA (tracrRNA) (100 nM) were incubated for 5 min at 95 ◦C, according to the manufacturer's instructions. Cas9 (100 nM), gRNA, and 1 × NEBu ffer 3 (New England Biolabs Inc, NEB) were incubated for 10 min at 25 ◦C to form the RNP complex. Amplified PCR products (300 ng) were then incubated for 60 min at 37 ◦C with the RNP complex. Proteinase K (800U/mL) was added to stop the reaction. The products were visualized using 1% agarose gel electrophoresis [13].

### *2.2. Maize Protoplast Isolation and Fluorescent Transfection Assay*

Mesophyll protoplasts were isolated following the protocol described by Sheen et al. [21] with some modifications. Etiolated maize seedlings were grown in vermiculite after disinfestation of the seeds with 70% alcohol (60 s), NaOCl2% (twice of 15 min), and triple washing with sterile water. Ten-days-old seedlings were used (three days under 16 h light/day and seven days in darkness). The middle portion of the second leaves were cut into thin strips (0.5–1 mm), and immersed in a cell-wall digestion enzyme solution (0.3% macerozyme R-10, 1.5% cellulase R-10, 10 mM of MES pH 5.7, 0.6 M mannitol, 10 mM CaCl2, 5 mM ß-mercapto, 0.1% BSA). The material was left in a vacuum for 30 min and gentle shaking at 40 rpm in the dark for 4 h. The protoplasts were released thoroughly by shaking at 80 rpm for 5 min After digestion, the protoplasts were diluted with the same volume of cold W5 solution [2 mM MES (pH 5.7), 154 mM NaCl, 125 mM CaCl2, 5 mM KCl] and filtered through a double filter (14 and 40 μM Nylon mesh). Protoplasts were collected after centrifugation at 100 g for 3 min and washed two times in 10 mL of W5 solution. Protoplasts were resuspended in cold MMG solution [0.4 M mannitol, 4 mM MES (pH 5.7), 15 mM MgCl2]. Its viability and concentration were determined using Fluorescein Diacetate (FDA; 0.2%) dye in the hemocytometer. To confirm the internalization of the RNP complex inside cells, an assay was performed using fluorescent-labeled tracrRNA molecules (ATTO 550, IDT) [9]. Microphotographs were taken using an inverted optic microscope ix80 Olympus.
