Plant genetic engineering stands to benefit greatly from the editing precision of CRISPR-Cas genome editing. However, introducing the workhorses of CRISPR into model and crop species, either with CRISPR plasmids or RNPs, is hindered by the molecular delivery challenge across the plant cell wall. The cell wall presents a rigid barrier to biomolecule delivery which is typically overcome by Agrobacterium or biolistic particle bombardment, the former of which is only tractable for DNA delivery. The success of both techniques is limited by host species, tissue type, and random transgene integration often resulting in constitutive transgene expression. Furthermore, many crop species are monocots and/or recalcitrant to tissue culture regeneration, which renders Agrobacterium and particle bombardment ineffective. In the case of CRISPR-Cas gene editing, transgene integration of the CRISPR components compromises genome stability, and segregation of the transgene requires laborious amounts of progeny screening. Furthermore, regulatory barriers in the United States inhibit transgenic plants from being swiftly brought to market. Nanomaterials offer a unique alternative to the current workhorses of plant genetic engineering due to their ability to passively traverse the cell wall and plasma membrane, selectively localize in tissues and organelles, and diversity of cargo loading capabilities. We present nanoparticle-mediated delivery and transient expression of CRISPR-Cas DNA vectors to leaf tissue. The delivery tool, a carbon nanotube-polyamine conjugate (PEI-SWNT), can be loaded with up to g/L amounts of DNA cargo and is administered by standard aqueous infiltration to the leaf abaxis. In Nicotiana benthamiana, we report preliminary results of 7.8 % indel efficiency, as measured by TIDE, of a GFP reporter transgene via PEI-SWNT delivery of a plasmid encoding CaMV 35S:Cas9 and AtU6:sgRNA. Notably, this system demonstrates similar expression efficiency to Agrobacterium but without transgene integration. Furthermore, we have demonstrated success in both dicots and monocots using PEI-SWNT delivery of DNA plasmids. We also discuss the application of PEI-SWNTs to transient expression in root tissue, and application of similar nanomaterial systems for DNA-free genome editing through delivery of CRISPR-Cas9 ribonucleoprotein (RNP).

Transient expression of CRISPR-Cas systems to mature plant tissues with nanoparticle-mediated delivery

2019 Fall National Meeting

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