Abstract
As important genome editing tools, CRISPR/Cas systems, especially those based on type II Cas9 and type V Cas12a, are widely used in genetic and metabolic engineering of bacteria. However, the intrinsic toxicity of Cas9 and Cas12a-mediated CRISPR/Cas tools can lead to cell death in some strains, which led to the development of endogenous type I and III CRISPR/Cas systems. However, these systems are hindered by complicated development and limited applications. Thus, further development and optimization of CRISPR/Cas systems is needed. Here, we briefly summarize the mechanisms of different types of CRISPR/Cas systems as genetic manipulation tools and compare their features to provide a reference for selecting different CRISPR/Cas tools. Then, we show the use of CRISPR/Cas technology for bacterial strain evolution and metabolic engineering, including genome editing, gene expression regulation and the base editor tool. Finally, we offer a view of future directions for bacterial CRISPR/Cas technology.
Highlights
Bacteria have rapid reproduction rates, are metabolically diverse, and can produce complex molecules that cannot be produced through conventional chemical syntheses, such as enzymes and a myriad secondary metabolites [1]
These approaches require extensive genetic modification of bacteria, which relies on the availability of robust genetic engineering tools
The emergence of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein (Cas) systems provided a number of new tools for genetic modification of bacteria
Summary
Bacteria have rapid reproduction rates, are metabolically diverse, and can produce complex molecules that cannot be produced through conventional chemical syntheses, such as enzymes and a myriad secondary metabolites [1]. A CRISPR/Cas system for Streptomyces was designed as a rapid multiplex genome editing tool, enabling targeted chromosomal deletions of 20 to 30 kb, with efficiencies from 70 to 100% [42]. Researchers used CRISPR/Cas9 technology to induce DSBs in actinomycetes and repaired the resulting blunt ends using the error-prone NHEJ pathway, resulting in insertions or deletions at the target site [58].
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