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Gene knock-in plays a crucial role in drug development. It is used in target validation by introducing specific genes into cell lines or animal models to confirm drug target efficacy. It also aids in establishing disease models, testing drug efficacy and safety in these models, and supporting drug screening through high-throughput screening in knock-in cell lines to identify potential drug candidates. Additionally, gene knock-in helps uncover drug mechanisms, optimize drug structure, and improve dosing strategies, expediting drug development while enhancing efficacy and safety.

Gene knock-in technology involves inserting an exogenous gene sequence into a specific within the genome for gene function studies or disease treatment. Edigene utilizes advanced gene editing tools, such as the CRISPR/Cas9 system, to guide nucleases to cut the target DNA, and employs homology-directed repair or non-homologous end joining to accurately insert the gene at the desired , achieving efficient and precise gene knock-in.

EDITGENE’s advantages in gene knock-in technology include: Guaranteed results: With 10 years of CRISPR gene editing experience and a team of PhDs from world-renowned institutions offering one-on-one support. High precision: EDITGENE’s optimized tools reduce off-target effects, enhancing editing accuracy. High efficiency: EDITGENE’s technology platform improves knock-in success rates, accelerating experimental progress. Customized service: Tailored knock-in solutions to meet specific research or therapeutic goals.

EDITGENE brings 10 years of CRISPR-based cell editing experience and offers one-on-one support from a team of PhDs from globally recognized institutions.

The main difference lies in the duration and stability of gene expression: Transient cell line – The target gene is expressed temporarily in cells, typically lasting hours to days, and is suitable for short-term experiments. Stable cell line – The target gene is stably integrated into the cell genome, allowing long-term expression, suitable for extended research and production.

Monoclonal screening is the process of isolating a single clone from a mixed pool of cells and expanding that clone into a cell line. Monoclonal screening ensures that the cell lines used originate from a single cell, guaranteeing a high degree of genetic background consistency. After cells are gene-edited or genetically modified, the genetic background differences among the cells in the initial cell pool can be significant, making subsequent experimental results inaccurate. By using monoclonal screening, researchers can obtain cell populations with consistent genetic backgrounds and stable gene edits, allowing for stable and accurate monitoring of phenotypic changes.

EDITGENE utilizes industry-leading 3D single-cell printing technology, which enables precise isolation and positioning of individual cells, significantly increasing the success rate and efficiency of monoclonal screening. This technology is widely applied in biomedicine research, antibody development, drug screening, and therapeutic selection, showcasing broad application prospects in cell research.

EDITGENE’s 3D single-cell printing technology employs non-contact operation, avoiding mechanical damage and background contamination, which helps maintain cell integrity and biological activity. This technology also minimizes human error in the traditional limited dilution method of monoclonal selection, ensuring the reliability of screening results.

Cell selection can follow these principles:
1.It should align with the research objectives.
2.The genes targeted by the sgRNA library should correspond to the cell's lineage.
3.The cells should be capable of stable passaging.
4.The transfection efficiency should be high.
5.Avoid primary cells whenever possible. Primary cells cannot be stably passaged and may experience significant cell death during the library screening process, which can hinder experiment completion. If primary cells must be used for library screening, mitigating this risk can be achieved by lowering cell coverage and choosing a library with fewer gRNAs to minimize the cell pool size and shorten the experimental duration.

CRISPR libraries can be divided into whole-genome libraries and subgenomic libraries. If the goal is to perform screenings across the entire genome, a whole-genome library is the best choice. Such libraries typically contain sgRNAs targeting the entire genome. If the research focus is specific, such as targeting only particular gene families or specific signaling pathways, a subgenomic library can be chosen to reduce unnecessary screening workload and costs.

What is the difference between a single-plasmid system and a dual-plasmid system for library vectors? A single-plasmid system can achieve gene editing with one transfection, making construction relatively simple, but the larger plasmid size can lead to lower infection efficiency. In a dual-plasmid system, two vectors are used, each carrying either the Cas9 or sgRNA expression cassette. A stable Cas9 cell line is first constructed, and then the sgRNA library is transfected into this cell line. This approach has several advantages:
1.Increased Editing Efficiency: The independent and stable expression of Cas9 protein and sgRNA on different vectors enhances editing efficiency.
2.Flexibility: Vectors can be designed and constructed flexibly based on experimental needs, such as loading two sgRNA expression cassettes into one vector.
3.Increased Viral Titer: By splitting into two plasmids, the load on each plasmid is reduced, facilitating viral packaging and increasing yield and titer.
4.Increased Stability: Independently constructing a stable Cas9 cell line ensures that the Cas9 expression levels and editing efficiency in each cell are approximately the same, enhancing experimental accuracy.

Maintaining the activity of cell cultures is crucial. Cells should not be allowed to reach confluence for more than 24 hours. Frozen new cells can restore transfection activity. The optimal cell plating density varies for different cell types or applications; however, for adherent cells, a confluence of 70% to 90% or a density of 5×10^5 to 2×10^6 suspended cells/ml typically yields good transfection results. It is important to ensure that cells are not fully confluent or in a fixed phase during transfection.

CRISPR detection reagents:
1.The RPA isothermal amplification kit can be stored at -20°C for long-term storage.
2.Target plasmids can be stored at -20°C for long-term use.
3.Cas proteins are sensitive to repeated freeze-thaw cycles; it is recommended to aliquot into multiple tubes and store at -80°C, retrieving them as needed for experiments. For short-term use, they can be stored at -20°C.
4.crRNA is prone to degradation and should be stored at -80°C if not used in the short term.
5. Probes, being double-stranded DNA, are relatively stable and can be stored at -20°C.

Both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) targets can activate the trans-cleaving activity (also known as collateral cleavage) of Cas12a, similar to Cas12b. However, the efficiency differs: ssDNA targets activate Cas12b trans-cleaving activity more efficiently than dsDNA targets, while dsDNA targets activate Cas12a trans-cleaving activity more efficiently than ssDNA targets.

The main differences among Cas9, Cas12, and Cas13 lie in their action mechanisms:
· Cas12 is activated to cleave ssDNA trans-cleaving after binding with guide RNA and target DNA.
· Cas13 is activated to cleave ssRNA trans-cleaving after binding with guide RNA and target RNA.
· Cas9 has not been reported to exhibit trans-cleaving activity.