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Knock-in

Definition

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

Guided by a single-guide RNA (sgRNA), the Cas9 endonuclease induces a double-strand break (DSB) at a specific genomic locus. The cell then activates the homologous recombination–mediated repair pathway. In the presence of a highly homologous donor DNA template, the donor sequence is used to repair the break, enabling precise insertion of the target DNA fragment into the genome at the desired 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

Knock-In Vector Design: A suitable knock-in vector is first designed, such as a plasmid, viral vector, or RNA-guided genome editing construct carrying the target gene or modified sequence. Vector design takes into account target gene specificity, homology arm length, and selection of the appropriate genomic integration site.

Cell Transfection: The engineered knock-in vector is introduced into target cells using methods such as chemical transfection, electroporation, or viral transduction. Optimization of delivery conditions is critical for improving knock-in efficiency and maintaining cell viability.

Screening of Knock-In Cell Lines: Following transfection or transduction, cells are screened to identify those with successful gene integration. This is typically achieved using selectable markers such as antibiotic resistance genes, fluorescent reporters, or genetic screening assays.

Monoclonal Screening: Single-cell cloning is performed to ensure clonality, genetic uniformity, and stability of the knock-in cell line. Common methods include limiting dilution, flow cytometry–based single-cell sorting, or clone picking..

Monoclonal Verification: The established monoclonal knock-in cell lines are further validated to confirm correct gene insertion and functional expression. Verification methods may include PCR, sequencing, protein expression analysis, and functional assays.

Application

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Knock-in

Definition

CRISPR-Cas9 gene knock-in principle

Service Process

Application

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