**4. Discussion**

CRISPR-Cas9 technology is a powerful tool for plant breeding and research. While still evolving as a technology to determine the rules for gRNA design and the algorithms to predict target and 'off-target' sequences, CRISPR applications still rely on empirical results to test the performance of new systems [25]. Notably, gene-editing results outcomes are frequently species-dependent [26]. Therefore, a CRISPR platform for different species with a rapid and efficient evaluation protocol is needed before commercialization.

Our experiments demonstrated the suitability of the PEG-delivered CRISPR-Cas9 RNPs system for gene-editing screening in maize. We showed that high-efficiency gene-edited maize cells could be obtained using less time-consuming (15 days) and labor-intensive procedures (PCR, agarose gel electrophoresis, and Sanger sequencing). In addition, the advantage of our system in relation to the use of vectors is that it prevents the integration and expression of exogenous DNA sequences, isolating the e ffect of gene-editing modification and avoiding transgene-introgressed side e ffects. Although non-integrating plasmids could be transfected into plant cells to deliver programmable nucleases, transfected plasmids are degraded in cells by endogenous nucleases, and the resulting small DNA fragments can be inserted at both on-target and o ff-target sites in host cells (Kim et al. 2014) [27]. For example, Braatz et al. [28] performed whole-genome sequencing after the transfection of the expression construct CRISPR-Cas9 in *Brassica napus* and found that the transformation resulted in at least five independent insertions of the vector backbone sequences in the plant genome.
