*4.1. Ribonucleoprotein Delivery in Plants*

In the present report, we show a positive correlation between the time of exposure to RNPs and the e fficiency of site-directed mutagenesis in maize, as ascertained with Sanger sequencing. In previous reports referring to the use of RNPs in plant protoplasts (Table 3), the authors used one or more di fferent concentrations of RNPs and Cas9:gRNA ratios, but the e ffect of exposure time on mutation frequencies was not tested [29,30]. CRISPR RNPs were delivered to apple, grapevine, brassica sp., lettuce, tobacco, and rice plants at less or equal than 20 min exposure time and their e fficiencies ranged between 0.1 and 40% [12,13,31]. In petunia and wheat protoplasts exposure for 30 min granted 0.2 up to 45% efficiency [14,17]; thus, suggesting that time of exposure might not alone explain indel frequency in di fferent plant systems. In our system, when all other factors are maintained, exposure time consistently increased indel frequency for all three gRNA sequences tested (up to approx. seven-fold change increase).

The Cas9:gRNA ratio also influences target e fficiency in a species-specific manner. Three di fferent Cas9:gRNA ratios were tested in apple and grapevine protoplasts [13]. While the 1:1, 1:3, and 3:1 ratios did not di ffer in mutation frequency for grapevine (0.1%), the 1:1 and 3:1 ratios increased indel frequency in two (6.6 and 2.6 fold change respectively) out of three gRNA sequences in apple. Overall, the results obtained for the 3:1 ratio are equivalent to our results applying the same ratio (from 3.3% up to 6.7% e fficiency). Cas9 concentration has been shown to be of major factor influencing the delivery of RNPs to plant cells. Woo et al. [12] tested 20 and 60 ug of Cas9 to *Arabidopsis* protoplasts and found that the editing e fficiency was not directly related to the Cas9 concentration but also dependent on the time course of the analysis. At 24 h after delivery, more e fficiency was observed when applying 20 instead of 60 ug of Cas9 (71 in contrast to 54%). Opposite results were obtained at 72 h after delivery. Nevertheless, increasing the amount of Cas9 (7.5, 15. 30, and 60 ug) was consistent with a crescent indel frequency in brassica sp. protoplasts [31]. The e fficiency results obtained in our study were similar to those obtained applying approximately 60–90 ug of Cas9, thus, indicating that a lower amount of Cas9 (45 ug) but a higher exposure time (40 min) might have similar cleavage levels. Overall, it is clear that the limited amount of studies investigating the RNP delivery into plant cells is insu fficient to draw definitive conclusions for increasing gene-editing e fficiency using this system.


**Table 3.** Publications with DNA-free gene editing in plants using CRISPR-Cas9 RNPs and other delivery methods for maize.


**Table 3.** *Cont.*

Note: N.A is 'not applicable' because transgenic plants were either selected using antibiotic marker genes or the analysis was not performed.
