Abstract
Genetic and molecular modifications of the large dsDNA chloroviruses, with genomes of 290 to 370 kb, would expedite studies to elucidate the functions of both identified and unidentified virus-encoded proteins. These plaque-forming viruses replicate in certain unicellular, eukaryotic chlorella-like green algae. However, to date, only a few of these algal species and virtually none of their viruses have been genetically manipulated due to lack of practical methods for genetic transformation and genome editing. Attempts at using Agrobacterium-mediated transfection of chlorovirus host Chlorella variabilis NC64A with a specially-designed binary vector resulted in successful transgenic cell selection based on expression of a hygromycin-resistance gene, initial expression of a green fluorescence gene and demonstration of integration of Agrobacterium T-DNA. However, expression of the integrated genes was soon lost. To develop gene editing tools for modifying specific chlorovirus CA-4B genes using preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs), we tested multiple methods for delivery of Cas9/sgRNA RNP complexes into infected cells including cell wall-degrading enzymes, electroporation, silicon carbide (SiC) whiskers, and cell-penetrating peptides (CPPs). In one experiment two independent virus mutants were isolated from macerozyme-treated NC64A cells incubated with Cas9/sgRNA RNPs targeting virus CA-4B-encoded gene 034r, which encodes a glycosyltransferase. Analysis of DNA sequences from the two mutant viruses showed highly targeted nucleotide sequence modifications in the 034r gene of each virus that were fully consistent with Cas9/RNP-directed gene editing. However, in ten subsequent experiments, we were unable to duplicate these results and therefore unable to achieve a reliable system to genetically edit chloroviruses. Nonetheless, these observations provide strong initial suggestions that Cas9/RNPs may function to promote editing of the chlorovirus genome, and that further experimentation is warranted and worthwhile.
Highlights
Research examining chloroviruses has provided many unexpected findings and concepts to the scientific community over the past 40 years [1]
Prior to testing the ability of Agrobacterium tumefaciens to genetically transform NC64A, experiments were performed to determine the concentrations of cefotaxime needed to rid cultures of NC64A of Agrobacterium following algal/bacterial co-cultivations and the level of hygromycin needed to kill all NC64A cells not integrating Agrobacterium T-DNA containing the hygromycin-resistance gene into their genome
Growth of A. tumefaciens was inhibited at a cefotaxime concentration of 100 mg/L (Fig 1A) whereas the growth of NC64A was found to be uninhibited in cefotaxime-supplemented media at the same concentration up to at least 1000 mg/L (Fig 1B)
Summary
Research examining chloroviruses has provided many unexpected findings and concepts to the scientific community over the past 40 years [1]. Despite these major achievements, no transformation system has been developed that allows the genetic modification of the large dsDNA viruses that infect certain unicellular Chlorella-like green microalgae. This transformation bottleneck creates a significant handicap in exploration of chlorovirus genomes that range from 290 to 370 kb and that encode many unidentified proteins. With the advent of CRISPR technology and the ongoing discovery of new giant viruses and their annotated genomes, we are armed with resources that have yet to be married
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