Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing

Abstract

The ability to carry out 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 and are present in freshwater environments throughout the world. 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. In an effort to develop gene editing tools for modifying specific chlorovirus CA-4B genes using preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs), we first 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). Agrobacterium -mediated transfection of chlorovirus host Chlorella variabilis NC64A with a binary vector containing a chlorovirus-encoded glycosyltransferase mutant gene was also examined. Attempts at developing a reliable chlorovirus transformation system were unsuccessful. However, in one experiment two independent virus mutants were isolated from macerozyme-treated NC64A cells incubated with Cas9/sgRNA RNPs targeting CA-4B-encoded gene 034r, which encodes a putative glycosyltransferase. Selection of these mutants using antibodies was dependent on a specific change in the pattern of glycans attached to the virus major capsid protein (MCP). 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, 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.