1. Technical Details
CRISPR/Cas9 system is a versatile tool for gene modification of nearly all types of cells. crRNA (CRISPR-derived RNA) binds with tracr-RNA (trans-activating RNA) by base pairing to form tracrRNA/crRNA complex, which guides the Cas nuclease to cleave and introduce sequence-specific double stranded DNA breaks (DSB) . By designing tracrRNA and crRNA, we can transform them into sgRNA, which is a synthetic RNA sequence that could guide Cas9 to bind and splice the targeted sequence of DNA.
Cas9 nuclease has two domains, NHN (cutting the complementary strand of crRNA) and RuvC (cutting the non-complementary strand of crRNA), which could cleave two single strands of DNA respectively and result in double stranded DNA breaks (DSB) . To repair the DSB, the cell uses its own DNA repair machinery to add or delete or replace pieces of DNA sequence via Homology Directed Repair (HDR) or Non-Homologous End Joining (NHEJ).
When cells use NHEJ to repair DSB, errors are more likely to occur. Deletion or addition of bases results in frameshift mutation and thus causes the target gene knockout.
2. Key Features
① Plasmid electroporation was applied to generate stable knockout cell lines. Multiple gRNAs were transfected to target a larger-segment and achieve a more complete knockout of the gene.
② Plasmid electroporation is transient expression, i.e. the plasmid will be degraded in the cell. Compared with lentivirus-based method, Plasmid electroporation avoid the risk that virus backbones randomly integrate with cell genome.
③ To provide a better and more comprehensive service, Ubigene can provide additional testing services, such as QPCR, Western Blot, etc.
④ Strict preliminary experiments will be carried out to increase the chance of success and greatly shorten the turnaround. Such experiments include monoclonal formation rate verification, optimal electroporation condition confirmation, minimum lethal concentration testing, target sites validation, etc.
① Vectors will be verified by restriction digestion, colony PCR and sequencing.
② The concentration of plasmid for electroporation must be greater than 1 ug/ul.
③ Positive clones were verified by PCR and sequencing. For cells with inconsistent allele, TA cloning will be carried out.
① To study the function of genes/proteins;
② To simulate human diseases for drug research and screening;
③ To verify targets, etc.
5. Cell lines that Ubigene had successfully modified their genome:
Human Colon Cancer Cell Line (HCT116)
Human Colon Cancer Cell Line (SW480)
Human Colon Cancer Cell Line (HT-29)
Human colon adenocarcinoma Cell Line
Human Hepatoma Cell Line (Hep3B)
Human Hepatocellular Carcinoma Cell Line
Rat Hepatoma Cell Line
Human Primary Colon Cancer Cell Lines
Human Gastric Cancer Cell Line (HGC-27)
Human Esophageal Squamous Carcinoma Cell Line
Human Esophageal Squamous Carcinoma Cell Line
Porcine small intestinal epithelial cell line
Human Breast Cancer Cell Line (MDA-MB-231)
Rat Breast Cancer Cell Line (4T1)
Human Pancreatic Carcinoma Cell Line (PANC-1)
Human Pancreatic Carcinoma Tumor Cell Line
Mouse Pancreatic Carcinoma Cell Line (PANC-1)
Human Prostate Cancer Tumor Cell Line
Human Prostate Cancer Cell Line
Mouse Acinar Pancreatic Cell Line (266-6)
Human Lung Cancer Cell Line (A549)
Human Lung Cancer Cell Line (Epithelium)
Human Non-small Cell Lung Carcinoma Cell Line (H1299)
Human Lung Adenocarcinoma Cell Line
Human Bronchial Epithelial Cell Line (BEAS-2B)
Human Bronchial Epithelial Cell Line
human embryonic lung fibroblasts
Mouse Macrophage Cell Line (RAW264.7)
Human Monocytic Cell Line (THP-1)
Human Erythroleukemia Cell Line
Human Leukemic Cell Line
Mouse Acute Myeloid Leukemia Cell Line
Mouse Myeloid Progenitor Cell Line
Human Bladder Cancer Cell Line
Human Adrenal Cortical Adenocarcinoma Cell Line
Mouse Mesangial Polyoma Cell Line
Rat Glomerular Mesangial Cell Line
Human Kidney Cell Line
Rhesus Monkey Kidney Cell Line
Human Renal Carcinoma CellLine
Human Embryonic Kidney Cell Line(HEK293)
Human Embryonic Kidney Cell Line(derived from 293)
Human Cervical Carcinoma Cell Line (HeLa)
Human Ovarian Adenocarcinoma Cell Line
C57BL/6 Mouse Embryonic Stem Cells
Mouse Embryonic Osteoblast Precursor Cells
Mouse Embryonic Fibroblasts (Preadipocytes)
Mouse Embryonic Fibroblasts
Chinese hamster ovary cells (CHO)
Human Vulvar Leiomyosarcoma Cell Line
Rat Cardiac Myocytes (H9C2)
Human Coronary Artery Endothelial Cell line (HCAEC)
Mouse Myoblast Cell Line (C2C12)
Human Neuroblastoma Cell Line
Mouse Neuroblastoma Cell Line
Human Glioblastoma Cell Line (U251)
Rat Glioblastoma Cell Line (C6)
Human Glioblastoma Cell Line
Human Glial Cell Line
Human Osteosarcoma Cell Line (MG63)
Human Osteosarcoma Cell Line
Immortalized Human Epidermal Cell Line
Mouse Chondrocyte Progenitor Cell Line
Human Malignant Melanoma Cell Line
Rat Muller Cell Line (rmc-1)
Human Tongue Squamous Carcinoma Cell Line
Human Nasopharyngeal Carcinoma Cell Line (C666-1)
Human Nasopharyngeal Carcinoma Cell Line (cne2z)
Mouse Squamous Cell Carcinoma Cell Line
Mouse Tumor Cells
Human Fibrosarcoma Cell Line (HT1080)
Ewing's Sarcoma Cell Line