Emergent CRISPR–Cas-based technologies for engineering non-model bacteria

Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated proteins (Cas) technologies brought a transformative change in the way bacterial genomes are edited, and a plethora of studies contributed to developing multiple tools based on these approaches. Prokaryotic biotechnology benefited from the implementation of such genome engineering strategies, with an increasing number of non-model bacterial species becoming genetically tractable. In this review, we summarize the recent trends in engineering non-model microbes using CRISPR–Cas technologies, discussing their potential in supporting cell factory design towards biotechnological applications. These efforts include, among other examples, genome modifications as well as tunable transcriptional regulation (both positive and negative). Moreover, we examine how CRISPR–Cas toolkits for engineering non-model organisms enabled the exploitation of emergent biotechnological processes (e.g. native and synthetic assimilation of one-carbon substrates). Finally, we discuss our slant on the future of bacterial genome engineering for domesticating non-model organisms in light of the most recent advances in the ever-expanding CRISPR–Cas field.