Pilot study: Novel CRISPR-Cas13 System Focusing on Viral Replication and Localization

Abstract

The flow of modular biology that influence how evolution act on clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) is difficult to understand. Mostly we must look at data to make interpretation about trends that can be used to infer the effect of selection pressures. However, in the big picture there is numerous determinants that is missing without trace. To fully understand the safe place of CRISPR-Cas in the ecological context, we must study the flow of mobile genetic elements (MGE) involved in complex fitness scenarios. For example, the effect of plant viruses and their insects' vectors on antiviral CRISPR-Cas applications. In basic research CRISPR-Cas can be used to investigate plant viruses in single cell protoplasts, plants or in the crude extracts of plant tissue. The cutting edge of CRISPR-Cas is the property of focus and the sensitivity that is needed to make competitive advances follows from the ability of accuracy and precession to make headway in the research of plant viruses. Uncontrolled flow of MGE can jeopardize the advantages of CRISPR-Cas cutting-edge over plant viruses. That risk needs to be localized to prevent plant viruses or their subviral relatives from developing resistance to CRISPR-Cas. Protoplasts may be regenerated into whole plants, which opens up the possibility of a new venue for virus-free plant materials. The work presented here is focused on CRISPR-Cas, plant viruses, plant material and selected aspects of plant cells' protoplasts as a bioassay including the characterization of subcellular localization of cas13bt3 based gene technology and potential application in research of plant viruses.

PART 1 investigated a novel CRISPR-Cas effector of the recently discovered class 2 systems' type VI (Cas13) superfamily of Bt's. This subfamily of Cas13b is compact and the Cas13bt3 effector protein is the smallest to be further analyzed in detail using structural methods. The literature part places the Cas13bt3 effector protein in perspective of the prokaryote origin and repurposed application in modern gene editing and biosensing. I took the initiative to work with this protein soon after the first published article describing the Bt superfamily in 2022, and anticipated that there would follow several studies based on the Cas13bt3 protein. Consequently, we started using the protein in the laboratory for research purposes. The experimental part involves repurposing wild type Cas13bt3 and the mutant (deactivated) dCas13bt3 for transfection of plant cells.

PART 2 elaborated on selected characteristic of plant viruses in connection with infection. Combining previous work in bioinformatics and other literature to research the nature of single-stranded RNA viruses, the relationship between our empirical knowledge about proteins and viral transcription indicated current strengths and weaknesses that is a topic for further studies. To overcome plant viruses there is a plethora of options, but no definite roadmap can be made because the ecology encompass surprising leaps of faith between insect vectors and plants. It seems the future of plant virus management is a cross disciplinary field that likely stand to benefit from artificial protein folding.

PART 3 studied the plant materials of the tobacco model Nicotiana benthamiana that is susceptible to many viruses infecting crop plants. The literature part was centered around climate challenges of crucial importance to high salinity and plant homeostasis that involves understandings of the most basic principles of cells' protoplasts, which can be used in future studies. Single protoplasts are interesting at all levels because modern gene editing, including CRISPR-Cas based technology, together with high-throughput omics reinvented forward and reverse genetics. In the experimental part plants were grown and leaf tissue harvested to isolate vigorous protoplast for the purpose of macromolecular transfection. I used the protoplast bioassay to characterize the subcellular localization of a catalytically inactive Cas13bt3 fusion protein tagged with red fluorescent protein (RFP) that previously demonstrated viable labelling of different cellular compartments.