Gene knock-in refers to the insertion of a new gene or DNA sequence into a specific location in the genome. This can be achieved through homologous recombination, viral vectors, or CRISPR/Cas9. Homologous recombination-mediated gene knock-in requires the introduction of a modified DNA fragment containing the desired gene sequence into the target site; viral vectors, such as retroviruses or adeno-associated viruses (AAVs), can be used to deliver new genes into the genome; CRISPR/Cas9 can also be used to insert a gene by using a donor DNA template along with sgRNA and Cas9 enzyme.
CRISPR-Cas9 gene knock-in principle
Under the guidance of sgRNA, Cas9 endonuclease cuts the double-stranded DNA at a specific location to create a DNA gap, and then the organism initiates the homologous recombination repair pathway. When there is a highly homologous DNA template (Donor DNA), Donor DNA will be used as the template. Repair is carried out to achieve the purpose of introducing the target fragment into the genome at a specific location.
When repairing DSBs through HDR, the cell repair system uses an additional exogenous fragment that is homologous to both sides of the target DNA as a template to synthesize a gene sequence complementary to the homologous sequence at the DSB. Since the exogenous fragment carries the mutation or target gene to be knocked in, it is possible to accurately introduce point mutations into the genome or insert the target gene.
Service Process
Design knock-in vector: First, a suitable knock-in vector must be designed, including a plasmid, viral vector, or RNA-guided editing complex carrying the target gene or modified sequence. The design of the knock-in vector should take into account the specificity of the target gene and the selection of the knock-in site.
Transfection: The designed knock-in vector is introduced into the target cells through methods such as chemical transfection, electroporation, or viral transduction. Choosing the appropriate transfection method and conditions can improve knock-in efficiency and cell survival.
Screen knock-in cell line: After transfection or transfection, cells need to be screened to obtain knock-in cell lines. This is typically done using genetic markers, fluorescent markers, or antibiotic resistance genes to select cell clones harboring the target gene.
Monoclonal screening: After obtaining the knock-in cell line, single clone selection is required to ensure its purity and stability. Single clone selection can be performed using methods such as limiting dilution, flow cytometry, or dilution methods.
Monoclonal verification: Finally, the knock-in effect of the established knock-in cell line needs to be verified, including detecting the expression level of the target gene, the presence and function of the modified sequence, etc.
Application
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