Markerless Gene Editing in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Abstract

The advent of single cell genomics and the continued use of metagenomic profiling in diverse environments has exponentially increased the known diversity of life. The recovered and assembled genomes predict physiology, consortium interactions and gene function, but experimental validation of metabolisms and molecular pathways requires more directed approaches. Gene function–and the correlation between phenotype and genotype is most obviously studied with genetics, and it is therefore critical to develop techniques permitting rapid and facile strain construction. Many new and candidate archaeal lineages have recently been discovered, but experimental, genetic access to archaeal genomes is currently limited to a few model organisms. The results obtained from manipulating the genomes of these genetically-accessible organisms have already had profound effects on our understanding of archaeal physiology and information processing systems, and these continued studies also help resolve phylogenetic reconstruction of the tree of life. The hyperthermophilic, planktonic, marine heterotrophic archaeon Thermococcus kodakarensis, has emerged as an ideal genetic system with a suite of techniques available to add or delete encoded activities, or modify expression of genes in vivo. We outline here techniques to rapidly and markerlessly delete a single, or repetitively delete several, continuous sequences from the T. kodakarensis genome. Our procedure includes details on the construction of the plasmid DNA necessary for transformation that directs, via homologous recombination, integration into the genome, identification of strains that have incorporated plasmid sequences (termed intermediate strains), and confirmation of plasmid excision, leading to deletion of the target gene in final strains. Near identical procedures can be employed to modify, rather than delete, a genomic locus.

TFS and Spt4/5 accelerate transcription through archaeal histone‐based chromatin

“…The amount of full-length transcripts at 2 min on protein-free templates in the absence of either factor is set to 1.0. [Colour figure can be viewed at wileyonlinelibrary.com] genomic modifications (Santangelo et al, 2008a;Hileman and Santangelo, 2012;Farkas et al, 2013;Jäger et al, 2014;Gehring et al, 2017). Hundreds of genes have successfully been deleted/modified using an integration-excision-based homologous recombination system (Sato et al, 2005;Santangelo et al, 2010;Hileman and Santangelo, 2012;Gehring et al, 2017).…”

“…[Colour figure can be viewed at wileyonlinelibrary.com] genomic modifications (Santangelo et al, 2008a;Hileman and Santangelo, 2012;Farkas et al, 2013;Jäger et al, 2014;Gehring et al, 2017). Hundreds of genes have successfully been deleted/modified using an integration-excision-based homologous recombination system (Sato et al, 2005;Santangelo et al, 2010;Hileman and Santangelo, 2012;Gehring et al, 2017). Two excision events are possible, but only one event results in the modification or deletion of the target locus, and by analyzing the results of hundreds of individual excision events, a statistical definition of essentiality can be derived (Hileman and Santangelo, 2012).…”

“…Two excision events are possible, but only one event results in the modification or deletion of the target locus, and by analyzing the results of hundreds of individual excision events, a statistical definition of essentiality can be derived (Hileman and Santangelo, 2012). For most non-essential genes, the expected 1:1 ratio of excision events that result in restoration of the original genome versus deletion/modification of the target locus have been observed (Gehring et al, 2017). Using established techniques (Gehring et al, 2017), we exhaustively attempted to delete TK0533 (encoding TFS), Spt4 (TK1698) and Spt5 (TK1419) from the T. kodakarensis genome.…”

“…For most non-essential genes, the expected 1:1 ratio of excision events that result in restoration of the original genome versus deletion/modification of the target locus have been observed (Gehring et al, 2017). Using established techniques (Gehring et al, 2017), we exhaustively attempted to delete TK0533 (encoding TFS), Spt4 (TK1698) and Spt5 (TK1419) from the T. kodakarensis genome. Despite analyzing >200 individual excision events for each locus, no strains were recovered with the desired targeted deletions.…”

FttA is a CPSF73 homologue that terminates transcription in Archaea

“…We were able to generate a strain (termed TK1428D) encoding an His 6 -affinity and HA-epitope tagged FttA( Figure 4g ). Strain TK1428D growth was indistinguishable from the parental strain 52 , 53 and N-terminally tagged FttA was easily recovered directly from TK1428D cell lysates in large abundance ( Extended Data Figure 6 ). Proteins co-purifying with FttA from TK1428D were identified by MuDPIT 54 , 55 , returning only a small number of proteins ( Extended Data Figure 7 ) that have minimal inferred activity related to transcription and gene expression.…”