(676e) Conjugation of CRISPR Machinery to Carbon Nanotubes for DNA-Free Genetic Engineering of Plants

Introduction of CRISPR machinery to a host plant genome may be accomplished through delivery of plasmids encoding Cas9 and sgRNA, in vitro transcribed Cas9 mRNA and sgRNA, or recombinant 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. Notably, transgenic crops are tightly regulated in many countries, wherein these regulations present significant financial and social barriers to the success of transgenic crops. Furthermore, the necessity of tissue culture introduces further limitations, as many crops are recalcitrant towards regeneration. Thus, there exists a great need to engineer model and crop plant species using non-tissue culture based methods and in the absence of recombinant DNA that could introduce risk of host integration.

RNA and RNP delivery simplify the workflow of plant gene editing by circumventing the need for plasmid optimization and improve specificity by reduction of off-target cleavage. Currently, introducing Cas9 RNPs or Cas9 mRNA/sgRNA into plants is not easily addressed by current delivery technologies, as Agro is only amenable to DNA delivery and biolistics rely on protein dehydration onto the carrier surface 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. Prior work has shown that pristine and cationic polymer-functionalized carbon nanotubes (CNTs) act as efficient carriers of siRNA and DNA plasmids, respectively, for the delivery of active biomolecules to plants via aqueous infiltration to the leaf abaxis [1]. We now present the development of DNA-free CRISPR-CNT conjugates for mRNA/sgRNA and RNP delivery to mature plant tissue without biolistics. While RNA delivery proceeds through direct adsorption of active mRNA or sgRNA to the pristine CNT surface, protein delivery necessitates an intermediate ligand to promote Cas9 adsorption to CNTs [2]. We present the design and synthesis of modular peptoid ligands 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 discuss the characterization of these diverse conjugates and their potential for use in genome editing applications.

1. Demirer, G. S. et al. Nat. Nanotechnol. 2019, 14 (5), 456–464.

2. Demirer, G. S. et al. Science Advances 2020, 6 (26), eaaz0495.