Microhomology Mediated High-throughput Gene Inactivation Strategy for Hyperthermophilic Crenarchaeon Sulfolobus islandicus.

Microhomology Mediated High-throughput Gene Inactivation Strategy for Hyperthermophilic Crenarchaeon Sulfolobus islandicus. Appl Environ Microbiol. 2017 Oct 13;: Authors: Zhang C, Whitaker RJ Abstract Sulfolobus islandicus is rapidly emerging as a model system for studying the biology and evolution within the TACK lineage of the Archaeal domain. As the tree of life grows, identifying the cellular function of genes within this lineage will have significant impacts on our understanding of the evolution of Last Archaeal Eukaryote Common Ancestor (LEACA) and the differentiation of Archaea from Eukaryotes during the evolution of the modernday cell. To progress our understanding of this key Archaeal organism, we report a novel high-throughput method for targeted gene inactivation in S. islandicus through one-step microhomology-directed homologous recombination (HR). We validated the efficacy of this approach by systematically deleting 21 individual toxin-antitoxin gene pairs and its application to delete chromosomal regions as large as 50 kb. Sequence analysis of 96 ArgD(+) transformants showed that S. islandicus can effectively incorporate donor markers as short segments through HR in a continuous or discontinuous manner. We determined that the minimal size of homology allowing native argD marker replacement was as few as 10 bp whereas argD marker replacement was frequently observed when increasing the size of homology to 30-50 bp. The mi...
Source: Applied and Environmental Microbiology - Category: Microbiology Authors: Tags: Appl Environ Microbiol Source Type: research