Implementing CRISPR-Cas9 Genetic Tools in Extremely Thermophilic, Lignocellulose Degrading Caldicellulosiruptor Bescii

Caldicellulosiruptor bescii is an anaerobic extreme thermophile with optimal growth temperatures between 70-78°C. As a lignocellulose-degrading bacteria, C. bescii shows promise as a host organism for transforming lignocellulosic feedstocks into biofuels, producing a more sustainable source of liquid fuels. In recent years, C. bescii has been genetically modified to produce final products such as alcohols and carboxylic acids. Currently, genetic modification of C. bescii is slow and labor intensive, making it difficult to perform the genetic manipulations necessary for fine tuning the metabolic pathways of this organism for industrial scale processes. This project works towards introducing thermophilic CRISPR-Cas9 technology into Caldicellulosiruptor to manipulate its genome more effectively. The most widely used CRISPR-Cas9 system was isolated from Streptococcus pyogenes, a mesophile, whose proteins are unstable above 45°C. However, recent discoveries of CRISPR-Cas9 systems native to thermophiles with stability in vitro from 55 to 100°C has made this gene editing technology possible in other species such as C. bescii. Our work to date has focused on knocking-in GeoCas9, a Cas9 protein isolated from Geobacillus stearothermophilus and stable up to 70°C, into C. bescii. In this proof of concept, GeoCas9 will knock-out the restriction enzyme CbeI in C. bescii as it is integrated into the genome. Then a replicating plasmid containing guide RNA (sgRNA) and regions for homology directed repair will be used to direct GeoCas9 to cut and modify the lactate dehydrogenase (ldh) gene in C. bescii. This will eliminate lactate production in the mutant strain; lactate production can then be measured to determine the efficiency of GeoCas9 as a genome editing tool.