UC Berkeley

The implementation of nascent CRISPR-based gene reading and writing tools in plant systems has lagged relative to mammalian counterparts. Methods for direct delivery of pre-formed ribonucleoproteins (RNPs) mediated by nanocarriers such as peptides and polymers have been explored with limited success. In part, this is due to the plant cell wall, a unique barrier to exogenous biomolecule delivery absent in other cell types. As such, the dominant model for studying direct delivery of RNPs and RNP-nanoparticles are protoplasts - plant cells that have had their cell wall enzymatically degraded. However, insights gained from such studies are limited in their generalization across species and the ultimate usefulness of such a simplified model is a question of concern.

Here, we report on the results of our efforts to translate three preparation methods previously tested in mammalian cells for polymer- and peptide-based Cas9 and CasΦ RNP-nanoparticles to both plant protoplast and 7-day-old seedlings: i) non-covalent complexing with charged polymers, ii) non-covalent complexing, and iii) tyrosinase-mediated covalent conjugation of various relevant peptide motifs. We use next generation sequencing to evaluate the ability of these RNP-nanoparticles to generate edits in the PDS and PDS3 genes of model plant systems Nicotiana benthamiana and Arabidopsis thaliana respectively. In seedlings, we observe no editing and confirm the absence of successful delivery events using the quantitative microscopy tool DCIP. In protoplasts, the amphiphilic peptide A5K or covalent attachment of peptides had no positive effect on editing efficiencies. However, the inclusion of the anionic polymer polyglutamic acid significantly improved intended editing efficiencies in both species relative to plain RNPs without negatively impacting protoplast viability. Our results support previously published literature that suggests the mechanism of action is stabilization of the RNP rather than the polymer having orthogonal activity in vivo. We believe that this cheap, simple, and accessible method of stabilizing Cas9 RNPs can be easily adopted by others working on direct protein delivery to plant protoplasts to increase gene editing. Furthermore, we believe this multi-scale analysis provides helpful insight into the feasibility of translating nanocarriers methodologies from mammalian to plant systems.