*3.1. In Vitro Cleavage Assay*

Cleavage activity of gRNAs 1 to 5 was tested using 0.5 μg of crRNA and 1.5 μg of Cas9 enzyme to 300 ng of DNA. While all the designed gRNAs were able to cleave PCR products of the IPK gene in our study, the di fferent gRNA sequences varied in their cleavage e fficiency (Figure 2). gRNA2, 3, and 4 showed the highest activity and were, therefore, chosen for subsequent experiments on the transfection of maize protoplasts.


**Figure 2.** Schematic diagram of the Maize IPK gene locus with the guide RNAs (gRNAs) target sites. The in vitro CRISPR-Cas9 assay showing the original and the cleaved fragments of the IPK gene in maize that were submitted to the RNP complex with the crRNA1, 2, 3, 4, and 5. Note: WT = Wild Type (control).

### *3.2. Targeted Mutagenesis in Maize Using CRISPR-Cas9 Ribonucleoproteins*

Protoplasts viability was checked before and 1 h after transformation. The viability of protoplasts at the isolation step was on average 75% and on average more than 65% after transformation. Therefore, these protoplasts were considered stable after transformation, which would allow further cell culture propagation.

Frequently, results indicating low e fficiency of CRISPR-Cas9 editing using RNPs delivery cannot discriminate low transfection rates from poor DNA cleavage and repair activity. In order to overcome this limitation and confirm the internalization of Cas9-gRNA RNPs, we performed a fluorescent microscopy assay. Labeled tracrRNA molecules confirmed the internalization of the RNP complex (Figure 3). Although this is not a quantitative method, it showed that at least one-third of the labeled molecules were internalized.

**Figure 3.** Microscope photographs of maize protoplast cells transfected with polyethylene glycol (PEG)-mediated CRSIPR-Cas9 RNP. The RNP containing the ATTO550-conjugated trans-activating crispr RNA (tracrRNA) was transfected into maize protoplasts and cells were monitored under fluorescent microscopy to check the transfection efficiency. Comparing to the cells without CRISPR-Cas9 delivery (**a**—control sample), the red fluorescent signal was detected in CRISPR-Cas9 treated samples (**b** and **c**—right). Photographs were taken with white light (left side) and fluorescent light (right side). Blue arrows indicate the internalization of the RNP complex. Scale bars: 100 μm.

RNPs containing gRNAs 2, 3, and 4 were transfected into isolated protoplasts with PEG 4000 and the results are displayed as the percentage of indels detected at the cleavage site based on Sanger sequencing and analysis with the ICE software v.2 (Figure 4). The concentration of 45 ug of Cas9 and 15 ug of gRNA to 100 ul protoplasts in a 3:1 ratio resulted in the best cost-efficiency correlation. This result is also in agreemen<sup>t</sup> with previous reports on RNPs delivery into protoplasts, which ranged from 30–60 ug of Cas9 in a 1:1 and 3:1 ratio [12,13,24]. Exposure time was tested for all three gRNA sequences in 20- and 40-min of exposure. While a longer exposure time to the RNP complex led to a higher mutation index for all gRNAs tested, the increase in mutation rate was not consistent among all gRNA sequences (approx. seven-fold, three-fold, and one-fold for the gRNAs 2, 3, and 4, respectively). Deletions were shown more frequent than insertions in this model system. A higher insertion rate was only observed for gRNA 2 at 40 min time of exposure.

DNA sequences from the universal primer used to amplify the IPK gene in maize (876 bp) from treated samples were compared to the same fragment in negative controls (no RNP delivered). Other negative controls (Cas9 or gRNAs delivered alone) were also tested against the first negative control and showed no DNA changes. The obtained gene-edited sequences for each gRNA at 40 min of exposure are displayed in Figure 5. About six sequence variants were of major contribution in gene-editing efficiency for the three selected gRNA sequences.

Results on the percentage of mutated sequences (technology efficiency), the size of the DNA change (number of base pair change), the type of DNA change (deletions or insertions), and a theoretical knockout (KO) score are summarized in Table 2. The indel percentage at 20 min of exposure was, on average, 1.63% in contrast to 4.37% at 40 min-exposure time. Overall, gRNA 4 was most efficient and

consistent at both exposure time. Intriguingly, gRNA 2 showed the lowest efficiency at 20 min (0.85%) but the highest efficiency at 40 min (5.85%). The Knockout Score accounts for the reads containing an amino acid frameshift change or 21 + bp indel. Thus, indicating the contributing indels that are likely to result in a functional knockout of the targeted gene. In the present study, the KO score was, on average, 0.83% for the 20 min and 3.17% for the 40 min of exposure treatment, which suggests that the majority of indels were frameshift modifications. In addition, only one gRNA at one time point presented a single base pair change as the most frequent mutation (gRNA 2 with a −1 bp). Notably, a deletion of 19 bp was the most abundant DNA change for gRNA 3 (0.9%). Moreover, all other gRNAs and exposure times showed a 2 bp deletion as the most frequent DNA change. Overall, the DNA change ranged from −28 bp to +12 bp change.

**Figure 4.** Frequency of mutations in maize protoplasts generated by CRISPR-Cas9 and measured by the Inference of CRISPR Editing Software—ICE software v.2. Different gRNAs and exposure time of the protoplasts to the RNP complex are represented. Percentages of insertions and deletions are represented in dark grey and light grey, respectively.

**Table 2.** Mutation rates in *Zea mays* IPK gene target region produced by CRISPR-Cas RNP delivery and analyzed by Sanger sequencing and ICE software analysis.


Note: KO = Knockout.


**Figure 5.** Sequence distribution of most efficient mutations identified with the ICE software around the IPK gene target site in *Zea mays*. Edited sequences were obtained after CRISPR-Cas9 RNP transfection to maize protoplasts. Forty-five μg of Cas9 preassembled with 15 μg of each gRNA were used in the protoplast transformation. Different exposure times of the RNP complex with the protoplast are presented. Cut sites are represented by black vertical dotted lines, insertions are represented by 'N' and deletions by black horizontal dotted lines.
