Knockout(KO) Cell Lines: CRISPR-U™ Technology

Typical Timeline for CRISPR Gene Knockout

The timeline for a CRISPR gene knockout can vary depending on the organism and specific experimental setup, typically ranging from a few weeks to several months. Here’s a general breakdown of the process:

• gRNA Design and Plasmid Construction (1–2 weeks)
• Cell Line Transfection & Pool Screening (2–3 weeks)
• Single-Cell Cloning (2–4 weeks)
• Clone Validation (2–3 weeks)

With 12 years of expertise in gene editing, Ubigene expert team has optimized the entire workflow, reducing the KO cell generation timeline to as fast as 4 weeks — significantly accelerating your research progress. Connect with Ubigene's experts now to customize your exclusive KO cell line!

Gene knockout (KO) is typically permanent, especially when using genome-editing techniques like CRISPR-Cas9. Here's why:

• Mechanism: In gene knockout, a DNA double-strand break is introduced at a specific location in the gene. This break is repaired by the cell’s own repair mechanisms, which often result in insertions or deletions (indels) and introduce an early stop codon,that disrupt the gene's coding sequence, causing it to lose its function. These mutations are passed on to daughter cells when they divide.
• Permanent in Cell Lines: If you're generating a knockout in a cell line, the knockout is usually stable over time, as the altered DNA will be present in every cell, and the mutation is passed down with cell division. You can even create clonal populations where all cells carry the same knockout mutation.
• Permanent in Organisms: If you’re creating a knockout in an organism (such as mice or other model organisms), the gene disruption will be passed on through heredity if the knockout is done in germline cells (e.g., sperm or eggs), leading to permanent knockouts in offspring. In some cases, you might generate heterozygous knockouts first, and then breed them to produce homozygous knockouts for more complete disruption.

1. Molecular Confirmation Methods:

• a) PCR and Gel Electrophoresis
• b) Sequencing
• c) Southern Blotting
• d) Western Blotting (Protein Level Confirmation)
• e) qRT-PCR (Quantitative Reverse Transcription PCR

2. Functional Assays:

• a) Phenotypic Analysis
• b) Reporter Genes
• c) Flow Cytometry (for Knockout Cells)

3. Knockout Mouse/Model Organism Specific Verification:

• a) Breeding and Genotyping (for Mice or Other Organisms)
• b) Histological Analysis