There has always been a huge debate about the origin of giant viruses and their contribution to the Tree of Life, which led to multiple schools of thought by various experts in the field. My scientific interest and pursuit of giant viruses brought me to one of the pioneer laboratories in this field, directed by Dr. Chantal Abergel. Here, I worked with Hugo and Nadege to explore the possibilities of genetic manipulation of giant viruses and their host. Genetic engineering is one of the strongest tools to understand the functionality of different genes and their role in infection and host-virus interaction.

Since the serendipitous discovery of Acanthamoeba polyphaga mimivirus in 2003, the species of Acanthamoeba has been utilized to discover and propagate new giant viruses. At present, the number of such viruses has increased enormously leading to the genesis of a separate phylum known as Nucleocytoviricota. These viruses harbor gigantic genome where 70% of the genes are ORFans with unknown functions, making them intriguing organisms for novel mechanisms and pathways. Although RNA silencing has been utilized to speculate the function of the genes, there is a gap in successfully performing the genetic manipulation of giant viruses and their host.

In this paper, we have reported a detailed protocol employing the CRISPR/Cas9 system to modify the genome of Acanthamoeba castellani and nuclear giant viruses. Although A. castellani is a polyploid eukaryote with 25 alleles of every gene in the genome, we were able to knock out the gene of interest with 100 % efficiency. Cas9 generates a double-stranded break that is repaired by non-homologous end joining (NHEJ) creating small indels that need to be screened rigorously by genotyping for complete gene knockout. The use of polycistronic gRNA or multiple gRNA to target a single gene is expected to perform better in such cases. In the case of nuclear giant viruses, repair of double-stranded breaks can be done by NHEJ or HR thus generating marker-free viruses which are further screened by genotyping.

However, the technique could not be used for cytoplasmic giant viruses as they set apart their DNA in the viral factory, thus protecting it from the Cas9 effector protein. Therefore, for cytoplasmic giant viruses, homologous recombination (HR) is a better tool for genetic manipulation, which can also be used for nuclear giant viruses. We employed HR to successfully knock out or tag the gene from/in the genome. We have constructed a range of vectors with selection cassettes (comprised of markers for drug resistance along with the constitutive early promoters and 3’UTR from the virus of interest) with (for 3HA or 3FLAG tag) or without tag (for knockout). The specific homologous arms need to be cloned at both the termini of the cassette for homologous recombination. Once the recombinant cassette is transfected to A. castellani followed by infection with the virus, homologous recombination eventuates producing recombinant viruses mixed with wild-type progenies.  These virus needs to be propagated and screened for single progeny of mutant viruses. Moreover, tagged genes can also be utilized as bait proteins to pull down interacting components that might be associated with similar pathways or mechanisms. For the knockout of the essential genes, we developed a strategy to trans-complement the gene expression in amoeba to avoid the requirement of that gene. The limitation of this technique is the scarcity of selection markers for A. castellani (currently only two; nourseothricin and geneticin) that limits up to two modifications per genome and only one for essential gene knockout.

This work sets a strong background for reverse genetics in giant viruses and Acanthamoeba castellani, generating a high impact on the whole community working on these viruses. The study will also influence the work of researchers who are studying other amoeba-associated microorganisms like Legionella, Parachlamydia, etc. The future goal of this work is to incorporate giant viruses into a group of cutting-edge genetically tractable organisms that can form a ground for future genetics and cell biology to study host-virus interactions.