Comprehensive CRISPR Gene Editing Solutions to Power Your Research: KO Cell Lines, CRISPR Screen & More

• Designing the Guide RNA (gRNA): A guide RNA is designed to specifically target a location in the genome where the new DNA sequence will be inserted. This RNA sequence is complementary to the target DNA sequence, ensuring that Cas9 cuts the correct location in the genome.
• Cas9 Protein: The Cas9 protein, which is part of the CRISPR system, is responsible for making a double-strand break at the target location in the DNA.
• Donor DNA Template: To achieve the knock-in, a donor DNA template is introduced into the cell. This template contains the genetic sequence that will be inserted into the genome. The donor template typically has homology arms (sequences that match the regions adjacent to the target break) to guide the cell's repair machinery.
• Homology-Directed Repair (HDR): After Cas9 cuts the DNA, the cell uses its HDR mechanism to repair the break. If a donor template is provided, the cell incorporates the new genetic material from the donor DNA into the genome at the break site, which results in the insertion of the desired sequence.

• Gene Therapy: Knock-in CRISPR can be used to insert healthy copies of defective genes in patients with genetic disorders. For example, correcting mutations in the CFTR gene (responsible for cystic fibrosis) by inserting a functional copy into a patient's cells.
• Disease Modeling: Researchers use knock-in to introduce specific mutations into the genome of animal models (like mice or rats) to study diseases or test therapies. This helps in creating models of genetic diseases such as cancer or neurodegenerative conditions.
• Synthetic Biology: Scientists can insert new genes into organisms (bacteria, yeast, or plants) to create new functions, such as engineered microbes that produce biofuels or therapeutic proteins.
• Drug Development: In drug discovery, knock-in CRISPR is used to create cell lines or animals that express specific mutations or diseases, allowing for more accurate testing of drugs or potential treatments.
• Cell Line Engineering: For industrial or therapeutic use, CRISPR knock-in can generate cell lines that express desired proteins, enzymes, or other bioactive molecules for research or commercial purposes.

• Cell Type: Choose an appropriate cell line or primary cell type for your experiment.
• Efficiency: CRISPR knock-ins generally have lower efficiency than knock-outs, and larger inserts can reduce efficiency further.
• Delivery Method: Choose a delivery method (electroporation, lipid-based, viral) based on your cell type's transfection efficiency.
• Homology-directed Repair (HDR): The success of knock-ins heavily relies on HDR, which is not as efficient as non-homologous end joining (NHEJ). You can enhance HDR by using HDR enhancers or small molecules (like SCR7).
• Verification: Always validate your knock-in using multiple methods (PCR, sequencing, protein expression).

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Validating CRISPR knock-in cells involves confirming:

• Correct insertion of the desired gene at the target location (via PCR, sequencing, Southern blot).
• Expression of the inserted gene at the RNA and protein levels (via RT-PCR, Western blot, fluorescence assays).
• Functionality of the inserted gene, especially for functional knock-ins (via fluorescence or activity assays).
• Absence of off-target effects, to ensure the edit is precise (via sequencing or specific assays).

After years of R&D of Ubigene, based on the original EZ-editor™ gene-editing technique, Ubigene has upgraded it to EZ-HRex™ New Technique. With the new technology, the proportion of HDR genotypes can reach up to 84%. Get in touch with our experts now

• Efficiency: The efficiency of knock-in can be low, especially compared to knock-out techniques, because homology-directed repair (HDR) is less common than the alternative repair pathway, non-homologous end joining (NHEJ), which often leads to deletions or errors rather than precise insertions.
• Off-target Effects: Although CRISPR technology is highly specific, there is still the potential for off-target edits, where the CRISPR machinery cuts DNA at unintended sites, leading to unwanted genetic changes.
• Delivery: Delivering the CRISPR/Cas9 components (guide RNA, Cas9, and donor DNA) into cells, especially in vivo (in living organisms), is challenging. Efficient delivery systems are crucial for the success of CRISPR knock-in applications.

Ubigene has revolutionized gene editing with the new EZ-HRex™ technique, now enhanced with the innovative U+ Molecule. This breakthrough allows HDR genotype proportions to reach up to 84% at the post-transfection cell pool level. Additionally, Ubigene offers 4 tailored solutions to address diverse mutation requirements, ensuring maximum flexibility and precision in your experiments! Contact us now

Ubigene offers global gene knock-in services, starting at just $6,480!