Abstract
The development of CRISPR-Cas9 based genetic manipulation tools represents a huge breakthrough in life sciences and has been stimulating research on metabolic engineering, synthetic biology, and systems biology. The CRISPR-Cas9 and its derivative tools are one of the best choices for precise genome editing, multiplexed genome editing, and reversible gene expression control in microorganisms. However, challenges remain for applying CRISPR-Cas9 in novel microorganisms, especially those industrial microorganism hosts that are intractable using traditional genetic manipulation tools. How to further extend CRISPR-Cas9 to these microorganisms is being an urgent matter. In this review, we first introduce the mechanism and application of CRISPR-Cas9, then discuss how to optimize CRISPR-Cas9 as genome editing tools, including but not limited to how to reduce off-target effects and Cas9 related toxicity, and how to increase on-target efficiency by optimizing crRNA and sgRNA design.