Organisms belonging to the Crenarchaeota lineage contain three PCNA subunits (proliferating cell nuclear antigen) while those in Euryarchaeota have only one as for Eukarya. To study the mechanism of archaeal sliding clamps, we sought to generate knockouts for each pcna gene in Sulfolobus islandicus, a hyperthermophilic crenarchaeon but failed with two conventional knockout methods. Then, a new knockout scheme denoted the marker insertion and target gene deletion (MID) was developed with which transformants were obtained for each pMID-pcna plasmid. We found that mutant cells persisted in transformant cultures during incubation of pMID-pcna3 and pMID-araS-pcna1 transformants under counter selection. Studying the propagation of mutant cells by semi-quantitative PCR analysis of the deleted target gene allele (Deltapcna1 or Deltapcna3) revealed that mutant cells lost propagativity, demonstrating that these pcna genes are absolutely required for host cell viability. Because the only prerequisite for this assay is to generate a MID transformant, this approach can be applied generally to any microorganisms proficient in homologous recombination.