Líffræðifélag Íslands - biologia.is
Líffræðiráðstefnan 2023
Höfundar / Authors: Björn Þór Aðalsteinsson (1), Hörður Guðmundsson (1), Hjördís Birna Árnadóttir (2), Þórdís Kristjánsdóttir (1), Antoine Moenaert (1), Cheryl Juanole (3), Ólafur Friðjónsson (1), Guðmundur Óli Hreggviðsson (1,2)
Starfsvettvangur / Affiliations: 1. Matís, Reykjavík, Iceland; 2. University of Iceland, Reykjavík, Iceland; 3. Université de Strasbourg, Strasbourg, France
Kynnir / Presenter: Björn Þór Aðalsteinsson
At Matís’s microbial biotechnology research group, we aim to develop practical applications for thermophilic bacteria. For the success of such endeavors, genome editing tools are generally necessary. Genome editing systems for thermophilic organisms are however relatively ill advanced – where available, their use is often labor intensive and time consuming – which slows progress in understanding and exploiting these diverse and interesting organisms.
We aimed to develop genome editing tools for thermophilic bacteria utilizing their endogenous CRISPR-Cas machinery. In Thermus thermophilus, Rhodothermus marinus and Thermoanaerobacterium strain AK17, we identified native CRISPR-Cas systems and predicted their protospacer adjacent motif (PAM) specificities. We constructed plasmid vectors that were designed to ‘hijack’ the native CRISPR-Cas systems such that their nuclease activities would be targeted towards the bacterial chromosome. When combined with a homologous recombination construct to direct gene knock-in or knock-out (KO), the vectors would enable genome editing and selection of edited clones through the action of the native CRISPR-Cas system. In T. thermophilus, the system was successfully used to generate several gene KO mutants at high efficiency. In AK17, application of the system led to generation of clones of mixed KO/WT genotype, and in R. marinus, no apparent CRISPR-Cas selection occurred.