Development of a CRISPR-Cas9 genome editing system for the extremely thermophilic bacterium Anaerocellum bescii

Anaerocellum (f. Caldicellulosiruptor) bescii is an extremely thermophilic (Topt 75-78 °C) anaerobic bacterium and is also a highly efficient degrader of lignocellulose, making it a promising platform for metabolic engineering. To date, chromosomal genetic manipulations in A. bescii have required multistep procedures involving homologous recombination with integrating vectors and counterselection. Here, we demonstrate the first implementation of CRISPR-Cas9 genome editing in A. bescii facilitated by a thermostable Cas9 enzyme from Geobacillus stearothermophilus (GeoCas9). Delivery of heterologous CRISPR machinery via a replicating vector enabled us to individually and sequentially delete genes for lactate dehydrogenase (ldh) and pyrE in A. bescii. We then deployed this system to delete a novel target, tryptophan synthase (trpBA), generating a tryptophan auxotrophic strain of A. bescii. Using this strain, we show trpBA complementation enables selection and maintenance of a replicating vector expressing a heterologous enzymatic reporter gene.