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Clustered regularly interspaced short palindromic repeats (CRISPR) technology is a powerful genome editing tool. Recently developed CRISPR-Cas12a system confers several advantages over CRISPR-Cas9, making it ideal for use in plant genome editing and crop improvement. While traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects, CRISPR-Cas12a delivered as ribonucleoproteins (RNPs) can effectively alleviate these potential issues. Here we present a detailed protocol for LbCas12a-mediated genome editing using RNP delivery in Citrus protoplasts. This protocol provides a comprehensive guideline for RNP component preparation, RNP complex assembly and delivery, and editing efficiency assessment., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Virus-like particles (VLPs) are genome-free protein shells assembled from virus coat proteins (CPs). The uniform and nanoscale structure of VLPs combined with their noninfectious nature have made them ideal candidates for the display of functional peptides. While the vast majority of VLPs are derived from spherical viruses, tobacco mosaic virus (TMV) produces a rod-shaped particle with a hollow central channel. However, under physiological conditions the TMV CP forms only disk-shaped macromolecules. Here, we describe the design, construction, purification, and processing of rod-shaped TMV-VLPs using a simple bacterial expression system. The robust nature of this system allows for the display of functional peptides and molecules on the outer surface of this novel VLP.

A modified version of the rod-shaped Tobacco Mosaic Virus (TMV1cys) provides a robust template for the self-assembly and fabrication of high-surface-area materials for numerous applications including batteries and sensors. TMV1cys surface fabrication is facilitated by the addition of a single cysteine residue to the virus coat protein that directs the vertical attachment of the virus particle onto substrate surfaces and enables deposition of functional inorganic layers. Here we describe the production and purification of the virus, its assembly onto suitable surfaces, and coating with metallic nickel and cobalt.