Site-specific knock-in refers to the technique of inserting exogenous functional genes into a cell's genome through homologous recombination with the homologous sequences in the genome. This process typically uses the CRISPR/Cas9 system, requiring the design of specific sgRNA and donor DNA.

Site-specific integration refers to the process of constructing an integration vector containing DNA fragments with homologous sequences. Through transformation, the exogenous DNA sequence undergoes homologous recombination with the target sequence, resulting in the targeted disruption of the target DNA sequence. A series of screening methods are then applied to obtain strains with the inserted gene.

1.What types of microbial gene knock-in are there?

Answer: There are two main types: in situ knock-in and site-specific knock-in. In situ knock-in refers to inserting a new gene at the site of the original gene knockout, which is the reverse process of gene knockout. In site-specific knock-in, regardless of the target gene's location, the gene is transferred to a precise insertion site using a transfer vector controlled by a specific promoter, ensuring accuracy in the insertion site.

2.What are the considerations for designing sgRNA?

Answer: The design of sgRNA affects the efficiency of gene knock-in. Key factors to consider include the sequence of the candidate editing site, its position, the orientation (positive or negative strand), GC content, and potential off-target sites.

3.How can the efficiency of microbial gene knock-in be improved?

Answer: To improve the efficiency and accuracy of gene knock-in, high-activity Cas9 protein variants and efficient gRNA designs should be selected. Additionally, optimizing the design of homologous arms and culture conditions can further enhance the success rate.