*4.2. CRISPR Delivery Methods in Maize*

Other delivery methods have been tested for maize as screening methods or gene-editing breeding methods, and these include PEG-mediated vector transfection, *Agrobacterium*-mediated, and biolistics (Table 3). The vast majority of studies still rely on vector-based transformation delivery of CRISPR. None of the listed studies have provided a cell-based screening method without the insertion of foreign vector-based DNA. On average, PEG-mediated vector transfection reached an average of about 12% efficiency, whereas *Agrobacterium* and biolistics reached 44% and 83%, respectively. In addition, most of these methods used embryogenic callus as explant material. Callus-based methods harbor chimeric tissues, thus, requiring subsequent genetic fixation to allow the stable inheritance of the edited traits. Therefore, these are not suitable material for genetic screening of successful gene-edited plants. Currently, many protocols are available for regeneration of whole plants from protoplasts. These include lettuce, tobacco, and rice, petunia, wheat, apple, and soybean. It is also suggested as a future choice for gene-edited maize and the list seems to expand because of the capabilities of the RNP technology [16,49]. The trade-off in efficiency percentage plays in return for avoiding unintended DNA integration and the potential undesirable biosafety risks.
