Endogenous CRISPR/Cas systems for genome engineering in the acetogens Acetobacterium woodii and Clostridium autoethanogenum .

Acetogenic bacteria can play a major role in achieving Net Zero through their ability to convert CO into industrially relevant chemicals and fuels. Full exploitation of this potential will be reliant on effective metabolic engineering tools, such as those based on the CRISPR/Cas9 system. However, attempts to introduce -containing vectors into were unsuccessful, most likely as a consequence of Cas9 nuclease toxicity and the presence of a recognition site for an endogenous restriction-modification (R-M) system in the gene. As an alternative, this study aims to facilitate the exploitation of CRISPR/Cas endogenous systems as genome engineering tools. Accordingly, a Python script was developed to automate the prediction of protospacer adjacent motif (PAM) sequences and used to identify PAM candidates of the Type I-B CRISPR/Cas system. The identified PAMs and the native leader sequence were characterized by interference assay and RT-qPCR, respectively. Expression of synthetic CRISPR arrays, consisting of the native leader sequence, direct repeats, and adequate spacer, along with an editing template for homologous recombination, successfully led to the creation of 300 bp and 354 bp in-frame deletions of and , respectively. To further validate the method, a 3.2 kb deletion of was also generated, as well as the knock-in of the fluorescence-activating and absorption-shifting tag (FAST) reporter gene at the locus. Homology arm length, cell density, and the amount of DNA used for transformation were found to significantly impact editing efficiencies. The devised workflow was subsequently applied to the Type I-B CRISPR/Cas system of , enabling the generation of a 561 bp in-frame deletion of with 100% editing efficiency. This is the first report of genome engineering of both and using their endogenous CRISPR/Cas systems.