Currently, CRISPR-Cas9-based site-specific integration is primarily used to modify and replace target genes in animal cells with human genes, thereby constructing animal (CDX) models and others.

The strategies to achieve Knock-In (KI) include: using synthetic oligonucleotides (Oligo) or single-stranded DNA (ssDNA) as donors for integrating large fragments. Using adeno-associated virus (AAV)-mediated delivery, which takes advantage of its property of existing in the cell nucleus in single-stranded form, to deliver ssDNA as a donor vector. And using double-stranded DNA fragments or circular plasmids as donors to achieve KI.

Figure 1. Service workflow for cell gene knock-in

Figure 2. Gene knock-in cell strategy

1.What is cell gene knock-in technology?

Answer: Cell gene knock-in is a technique that precisely inserts a specific exogenous gene fragment into the cell genome. This is typically achieved through advanced gene-editing tools, such as the CRISPR/Cas9 system combined with the homologous recombination repair mechanism. By designing guide RNA (gRNA) targeted at the insertion site, Cas9 nuclease induces a double-strand break at a specific location in the cell genome. A repair template containing the target gene fragment and sequences homologous to the target insertion site is provided. The cell uses homologous recombination repair to insert the gene fragment accurately into the target location.

2.What are the advantages of cell gene knock-in technology?

Answer: On one hand, it allows for precise insertion of the target gene at specific locations, enabling accurate modification of the cell genome. This facilitates gene function research, expression regulation, and protein-protein interactions. On the other hand, by knocking in genes with specific functions, such as reporter genes, tagged genes, or therapeutic genes, it provides powerful tools for cell biology research, disease model creation, and gene therapy.

3.What is the process for cell gene knock-in services?

Answer: The process includes cell testing, knock-in vector construction, donor vector construction, cell transfection (introducing gRNA, Cas9, and Donor into cells), constructing the gene mutation Cell pool, monoclonal screening and identification, and cultivating and verifying stable knock-in cell lines.