Tinker-Kulberg, R. | bioRxiv Science

bioRxiv2023

The "Duckweed Dip": Aquatic Spirodela polyrhiza Plants Can Efficiently Uptake Dissolved, DNA-Wrapped Carbon Nanotubes from Their Environment for Transient Gene Expression

Duckweeds (Lemnaceae) are aquatic non-grass monocots that are the smallest and fastest-growing flowering plants in the world. While having simplified morphologies, relatively small genomes, and many other ideal traits for emerging applications in plant biotechnology, duckweeds have been largely overlooked in this era of synthetic biology. Here, we report that Greater Duckweed (Spirodela polyrhiza), when simply incubated in a solution containing plasmid-wrapped carbon nanotubes (DNA-CNTs), can directly up-take the DNA-CNTs from their growth media with high efficiency and that transgenes encoded within the plasmids are expressed by the plants--without the usual need for large doses of nanomaterials or agrobacterium to be directly infiltrated into plant tissue. This process, called the "duckweed dip", represents a streamlined, hands-off tool for transgene delivery to a higher plant that we expect will enhance the throughput of duckweed engineering and help to realize duckweeds potential as a powerhouse for plant synthetic biology. (148 words)

bioengineering↗

bioRxiv2021

Polyvalent Guide RNAs for CRISPR Antivirals

CRISPR biotechnologies, where CRISPR effectors recognize and degrade specific nucleic acid targets that are complementary to their guide RNA (gRNA) cofactors, have been primarily used as a tool for precision gene editing1 but possess an emerging potential for novel antiviral diagnostics, prophylactics, and therapeutics.2-5 In gene editing applications, significant efforts are made to limit the natural tolerance of CRISPR effectors for nucleic acids with imperfect complementarity to their gRNAs in order to prevent degradation and mutation at unintended or "off-target" sites; here we exploit those tolerances to engineer gRNAs that are optimized to promote activity at multiple viral target sites, simultaneously, given that multiplexed targeting is a critical tactic for improving viral detection sensitivity,3 expanding recognition of clinical strain variants,6 and suppressing viral mutagenic escape from CRISPR antivirals.7 We demonstrate in vitro and in higher plants that single "polyvalent" gRNAs (pgRNAs) in complex with CRISPR effectors Cas9 or Cas13 can effectively degrade pairs of viral targets with significant sequence divergence (up to 40% nucleotide differences) that are prevalent in viral genomes. We find that CRISPR antivirals using pgRNAs can robustly suppress the propagation of plant RNA viruses, in vivo, better than those with a "monovalent" gRNA counterpart. These results represent a powerful new approach to gRNA design for antiviral applications that can be readily incorporated into current viral detection and therapeutic strategies, and highlight the need for specific approaches and tools that can address the differential requirements of precision gene editing vs. CRISPR antiviral applications in order to mature these promising biotechnologies.

bioengineering↗