Evolution of giant pandoravirus from small icosahedral viruses revealed by CRISPR/Cas9

Giant viruses (GVs) are a hotspot of unresolved controversies since their discovery, including the definition of “Virus” and the existence of a fourth domain of life 1-3 . While increasing knowledge of genome diversity has accumulated 4 , functional genomics was largely neglected. Here, we describe an experimental framework to genetically modify nuclear GVs and its host Acanthamoeba castellanii using CRISPR/Cas9, allowing us to uncover the evolution from small icosahedral viruses to amphora-shaped GVs. Ablation of the icosahedral major capsid protein in the evolutionary intermediate mollivirus highlights a stepwise transition in virion shape and size. We additionally demonstrate the existence of a reduced core essential genome in pandoravirus, reminiscent of their proposed smaller ancestors. Genetic expansion led to increased genome robustness, indicating selective pressures for adaptation to uncertain environments. Overall, we introduce new tools for manipulation of the unexplored genome of nuclear GVs and demonstrate that viral gigantism can arise as an emerging trait.