Construction of the Gene Tagging and Knock Out (GTKO) System for Reliable Genetic Analysis of Nuclear Genes in Cyanidioschyzon merolae

The unicellular red alga Cyanidioschyzon merolae is a eukaryotic photosynthetic model organism used for basic and applied cell biology studies. Its nuclear genome can be modified by homologous recombination with exogenously introduced DNA. The comparison of mutants with isogenic strains is critical for reliable genetic analyses; however, this has been impossible thus far. Strain construction and genetic complementation studies take a long time and give rise to unstable phenotypes. In this study, we modified the pMKT vector system described in Takemura et al. (2018) by adding an upstream sequence of the target gene, after the URA5.3T marker, to create a double sandwich of repeat elements at the target gene genomic loci after transformation. 5-fluoroorotic acid selection-based DNA excision yields two mutants simultaneously: an epitope-tagged mutant with only the URA5.3 marker eliminated; and a knockout mutant with both URA5.3T and the target gene's open reading frame eliminated. This system can provide multiple sets of isogenic controls in parallel, and further reveal the essentialness of a gene. Here, we introduce the multi-functional mutant design system Gene Tagging and Knock Out (GTKO), and demonstrate its utility by creating GTKO mutants of the CmBTB1 and CmBTB2 genes in C. merolae.