CRISPR Cas9 Gene Editing - CRISPR Library Screening - Gene Knockout Cell Line - Knock in Cell Line - Cas12a – EDITGENE

4.Poor cell condition after lentiviral infection may be caused by various factors. Here are some possible reasons and corresponding solutions: 1.High viral titer: High titers of lentivirus may cause cytotoxicity, preventing normal cell growth. Solution: Lower the viral titer and conduct a series of dilution experiments to find a titer that effectively transduces without adversely affecting cell growth. 2.Poor cell condition: The health status of cells before infection can affect growth after infection. Solution: Ensure cells are in optimal condition for infection, for example, by changing to fresh culture medium 24 hours before infection and ensuring appropriate cell density. 3.Toxicity of gene expression mediated by the virus: The gene carried by the lentiviral vector may be toxic to the cells, affecting their growth. Solution: If possible, use a control vector to determine if the problem is related to gene expression, and select appropriate vectors or genes for research. 4.Excessive antibiotic selection pressure: If antibiotics are used to select transfected cells, excessive concentrations of antibiotics may inhibit cell growth. Solution: Optimize the antibiotic concentration and use gradient experiments to determine the optimal concentration.

Traditional CRISPR/Cas9 technology achieves gene editing by introducing double-strand breaks at the target DNA site and then using the cell’s homologous recombination repair mechanism. This approach carries multiple risks, such as lower editing efficiency, reduced homozygous mutation rates, and random insertions or deletions. Prime Editing, however, does not require double-strand breaks. With its Cas9n-RT editing enzyme system and pegRNA, Prime Editing achieves more accurate and safer gene editing with reduced off-target effects.

EDITGENE’s newly upgraded seventh-generation Bingo™ Prime Editing (PE7) platform optimizes editing protein and RNA editing activity. Compared to the fifth-generation PE technology, point mutation success rates and gene editing efficiency have significantly improved, with one-on-one support from PhDs from globally renowned institutions.

EDITGENE’s Bingo™ Prime Editing 7 (PE7) platform is built upon over ten years of gene editing experience, with optimization and advancements derived from thousands of gene editing CRO projects, achieving significantly higher success rates than traditional site-specific mutation systems. The Bingo™ Prime platform utilizes highly efficient reverse transcriptase and precise guide RNA design, ensuring each point mutation reaches the desired outcome.

Prime Editing is a novel gene editing technology that enables precise gene editing without introducing double-strand DNA breaks. It has two core components: pegRNA and the PEmax gene-editing enzyme (Cas9n-RT). PegRNA not only targets the desired sequence but also contains the base modification information. In the editing system, pegRNA guides PEmax to the designated edit site, nicks the DNA single strand, and reverse transcribes the sequence within the pegRNA to modify, inserting it into the target genome location, thereby achieving precise single-base substitutions or small insertions and deletions

Not all genes are suitable for knockout. Some gene knockouts may result in cell death or severe dysfunction, particularly for essential genes. In such cases, conditional knockouts or gene knockdowns (e.g., RNAi) may be used instead.

Researchers use KO cell lines to investigate gene functions by observing the effects of gene deletion on cellular behavior. This helps in understanding the role of genes in various processes like cell growth, metabolism, and signal transduction. KO cell lines are vital for studying diseases like cancer, genetic disorders, and neurodegenerative diseases.

EDITGENE provides access to a comprehensive library of over 4,500 high-quality knockout (KO) cell lines, enabling researchers to save valuable time. Our custom gene knockout services are highly efficient, boasting a high positive rate while minimizing off-target effects. Clients also benefit from personalized, one-on-one support from a team of PhD experts from globally renowned institutions, ensuring top-tier service and results.

KO (Knockout) cell line is a cell line where a specific gene has been completely removed or rendered non-functional through gene editing technologies such as CRISPR-Cas9. These cell lines are critical for understanding gene functions and disease mechanisms.

KO cell lines can be applied to various cell types, including cancer cells, stem cells, and primary cells, but different cell types may have varying sensitivities to gene editing, and may vary among different cell types. In certain cell types, achieving gene knockout may require optimization of transfection conditions and selection of appropriate gene-editing tools.

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.

Gene knock-in technology involves inserting an exogenous gene sequence into a specific location 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 location, achieving efficient and precise gene knock-in.

iPSCs have broad clinical potential, including applications in cell therapy (e.g., for diabetes or heart disease treatment), tissue engineering (e.g., development of artificial skin or liver tissue), and personalized drug screening (e.g., selecting optimal treatments based on a patient’s specific cellular response). These applications may transform treatment methods, offering more effective and personalized medical services.

Both iPSCs and embryonic stem cells (ESCs) have pluripotency, but iPSCs are obtained by reprogramming the somatic cells, while ESCs come from early embryos. IPSC does not involve the use of embryos and does not violate ethical requirements, so it can also avoid immune rejection issues in scientific research. Therefore, iPSC is considered to be a preferred choice.

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.