UC Berkeley

Introducing CRISPR machinery to a host plant genome may be accomplished either through expression of recombinant plasmids or direct delivery of the Cas9 ribonucleoprotein (RNP). However, introducing the biomolecular workhorses of CRISPR into model and crop species is hindered by the molecular delivery challenge across the plant cell wall. The cell wall presents a rigid barrier to biomolecule delivery which can be overcome by Agrobacterium or biolistic particle bombardment, the success of which is limited by host species, tissue type, and random transgene integration. Furthermore, the necessity of tissue culture introduces further limitations, as many crops are recalcitrant towards regeneration. RNP delivery simplifies the workflow of plant gene editing by circumventing the need for plasmid optimization and improves specificity by reduction of off-target cleavage. However, introducing Cas9 RNPs into plants is not easily addressed by current delivery technologies, as Agrobacterium is only amenable to DNA delivery and biolistics rely on protein dehydration which can result in loss of Cas9 activity. Nanomaterials offer an addition to the workhorses of plant genetic engineering due to their ability to load diverse cargo, traverse the cell wall and plasma membrane, and selectively localize in tissues and organelles. We have developed a polyamine-carbon nanotube conjugate (PEI-SWNT) which is loaded with DNA and administered by aqueous infiltration to the leaf abaxis. In Nicotiana benthamiana, we report indel generation in a GFP transgene via PEI-SWNT plasmid delivery. We also present the development of Cas9-SWNT conjugates for RNP delivery to mature plant tissue without biolistics – both through direct binding of an engineered Cas9 variant to the CNT surface and through the inclusion of a noncovalent peptoid intermediate for binding of WT Cas9 to the CNT surface. Peptoids, or poly-N-substituted glycines, possess remarkable biostability and peptide-like structural properties. We present the design and synthesis of modular peptoids with two domains, a CNT-binding domain and an RNP-binding domain, and demonstrate screening of a peptoid library based on these domains for non-covalent binding of Cas9 to peptoid-CNTs. We have identified charge and the protein:SWNT ratio as key variables in the design of a stable conjugate, where a stable RNP-peptoid-conjugate can then enable cytosolic delivery of preassembled Cas9 RNP to mature plant cells. Lastly, we report indel generation in the model gene target phytoene desaturase (PDS) from N. benthamiana via peptoid-SWNT mediated delivery of Cas9 RNPs.