**5. Conclusions**

We have shown that RNPs could be used for targeted CRISPR-Cas9 via PEG delivery as a model system to screen for gene-editing outcomes in maize. Target insertion and deletion DNA changes (In/Dels) were induced using 45 ug of Cas9 and gRNA used at a 3:1 ratio, and a positive correlation between exposure time (20 and 40 min) and indel frequency was observed. By targeting preserved coding regions, we can anticipate that the model could be applied to several maize varieties by validation using IPK gene. However, in vivo In/Del frequencies differed among gRNA sequences. In addition, the proposed method for sequencing analysis is also restricted to a window of 30 bp deletions and 14 bp additions. Further studies will be focused on CRISPR-Cas9 off-target activity and on the regeneration of edited protoplasts.

**Author Contributions:** Conceptualization, R.O.N. and S.Z.A.-T.; Methodology, R.R.A.S., C.A.C. and S.Z.A.-T.; Software, R.R.A.S.; Validation, R.R.A.S. and C.A.C.; Formal Analysis, R.R.A.S., C.A.C. R.O.N. and S.Z.A.-T.; Investigation, R.R.A.S., C.A.C. and S.Z.A.-T.; Resources, R.O.N. and S.Z.A.-T.; Data Curation, R.R.A.S. and S.Z.A.-T.; Writing—Original Draft Preparation, R.R.A.S. and S.Z.A.; Writing—Review & Editing, R.R.A.S., C.A.C. R.O.N. and S.Z.A.-T.; Visualization, R.R.A.S. and S.Z.A.; Supervision, R.O.N. and S.Z.A.-T.; Project Administration, S.Z.A.-T.; Funding Acquisition, S.Z.A.-T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by GenØk Centre for Biosafety through its funds from The Ministry of Climate and Environment in Norway. Scholarship from National Council for Scientific and Technological Development (CNPq) was provided to R.R.A.S. and R.O.N.

**Acknowledgments:** We would like to thank Lilian Machado (UFSC) for taking care of the Sanger sequencing. The authors are also grateful to Vinicius Vilperte for insightful comments and suggestions on the sequencing analysis and also to Miguel Guerra for his support in using his laboratory. This work was financed by GenØk Centre for Biosafety through its funds from The Ministry of Climate and Environment in Norway, whose support is gratefully acknowledged.

**Conflicts of Interest:** The authors declare no conflict of interest.
