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

CRISPR-Cas9 genome editing was successfully implemented in the extremely thermophilic anaerobe Anaerocellum bescii (Topt 75–78°C), enabling sequential gene deletions.

• Why it matters: Chromosomal engineering in A. bescii previously required multistep homologous recombination with integrating vectors and counterselection, limiting metabolic engineering workflows.
• What they did: A thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) was delivered via a replicating vector to delete ldh and pyrE, then trpBA.
• The result: Deleting trpBA produced a tryptophan auxotrophic A. bescii strain, and trpBA complementation enabled selection and maintenance of a replicating vector expressing a heterologous enzymatic reporter gene.