**1. Introduction**

The development of technologies related to genetic improvement, such as transgenesis and more recently, genome editing, have changed the way humans have grown food for thousands of years. Today, the most promising tool to DNA manipulation is CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), a gene-editing technology that has been adapted from the bacterial immune system against viral infections [1]. Currently, a target site in the host genome can be reached by an endonuclease enzyme (i.e., Cas9) led by a guide RNA molecule (gRNA) that contains the target-specific

sequence. This protein complex forms the CRISPR-Cas9 ribonucleoproteins (RNPs) gene-editing system. Cas9 introduces a site-specific double-stranded DNA break (DSB) followed by the natural cell repair of disrupted genome integrity by error-prone non-homologous end-joining (NHEJ) or homology-directed repair (HDR) [2]. Therefore, this tool allows the in vivo modification of the DNA at the gene sequence of interest, with unprecedented speed, and has made it a milestone in manipulating and producing living modified organisms.

Although much is already known about the principles of CRISPR-Cas9 genome editing, the likelihood of different outcomes in terms of resolution, efficiency, accuracy, and DNA modification structure has shown to be species-dependent. Various factors, including target site choice, gRNA design, the properties of the endonuclease, the type of DSB introduced, whether or not the DSB is unique, the quantity of the endonuclease and gRNA, and the intrinsic differences in DNA repair pathways in different species, tissues, and cells will result in differences in the mutation signatures generated in plant species [3].

In the case of maize, most researchers acknowledge that *Zea mays* L. is an ancient amphidiploid species with a duplicated chromosome number of *n* = 10 [4]. Multiple independent domestication events have also contributed to the genetic variability encountered in modern maize [4]. In addition, the genetic variants and the underlying mechanisms influencing variance heterogeneity in maize have so far hidden additive genetic effects and epistatic interaction effects in elite varieties [5]. Therefore, CRISPR can be a useful tool to access genomic regions in the maize genome, which are difficult to achieve by conventional breeding.

While CRISPR technology has already been tested on commercial crops to increase yield, drought tolerance and growth under limited nutrient conditions, improve nutritional properties and develop resistance to plant pathogens [6]; breeding and research of major monocotyledon species, more specifically maize, are still at its infancy. Maize has shown to be an exemption in the plant portfolio for the in silico analysis of potential Cas9 target sites as only 29.5% of annotated transcripts matched a specific gRNA [7]. Among eight analyzed plant species, maize had the largest genome, the highest GC content, and the greatest number of annotated transcripts. Thus, reflecting the abundance of highly repetitive DNA and dispersed repeats, which may be challenging to develop unique target sites for the majority of genes in maize [7].

Despite such challenges, CRISPR technology opens up the possibility for genome changes without foreign introgression of DNA vectors. CRISPR-Cas9 technology can be used as RNP complexes without the introgression and expression of a transgenic cassette in the host genome [8]. Such an approach would avoid a number of generations of backcrossing, expression vectors, and other invasive methods of cell penetration (e.g., biolistics) that can lead to gene disruption, including large deletions, partial trisomy, genome shattering events, and plant mosaicism [9]. Overall, these side effects can mask or interfere in the target gene functional analysis, and further additional biosafety concerns prior to commercial release.

CRISPR RNPs can be delivered directly to plant cells without the cell wall. Therefore, prior to transfection, the cell wall must be removed through enzymatic digestion reactions. Protoplast cells are viable in vivo biological material for DNA-free CRISPR delivery in plants. In addition, protoplasts are viable after transfection, which allows further tissue cultivation and propagation.

Delivery of preassembled Cas9 protein-gRNA RNPs or plant DNA-free genome-editing techniques are not exempt from off-target effects but represent an approach in which the effects of Cas9 could be isolated from other more invasive techniques [8,10,11]. This approach was first demonstrated in *Arabidopsis thaliana*, tobacco, lettuce, and rice protoplasts, including the regeneration of gene-edited lettuce [12]. After that, a few successful attempts were also accomplished on grapevine and apple [13], *Petunia* × *hybrida* [14], potato [15], and on soybeans and tobacco using CRISPR-Cpf1 (CRISPR from *Prevotella* and *Francisella*), recently named Cas12a (for review, please read Metje-Sprink et al. [8]). Maize and wheat plants with targeted mutations have also been successfully obtained by delivering gold particles coated with the RNPs into embryo cells (biolistics), followed by post-bombardment culture

and plant regeneration [16]. However, the frequency of obtaining genome-edited plants was relatively low, since only 0.3–0.9% of regenerated maize plants possessed biallelic mutations [16].

The few studies published on maize genome editing rely mostly on the stable transformation [17–20]. In the manuscript, we delivered Cas9-gRNA RNP into maize leaf protoplasts via polyethylene glycol (PEG)-calcium mediated transfection, and indicated that In/Del mutations occurred with relatively high e fficiency of 0.85–5.85% among the PEG-calcium-treated protoplast. We targeted the *inositol phosphate kinase* gene (IPK) involved in the phytic acid biosynthetic pathway. To develop a standard protocol for di fferent maize varieties, we designed gRNAs and primers complementary to coding regions in exon three that are conserved in the species, in order to evaluate the e fficiency and spectrum of DNA changes generated by CRISPR-Cas9 technology in maize, and also add relevant information to the safety of gene-edited organisms. This e fficient and relatively easy assay method for the selection of gRNA suitable for editing of genes of interest will be highly useful for genome editing in maize, since the genome size and GC-content are large and high in the maize genome, respectively.
