Knockout Cell Lines

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CRISPR-U™ Cell Line
CRISPR-U™ (based on CRISPR/Cas9 technology), developed by Ubigene, is more efficient than general CRISPR/Cas9 in double-strand breaking, and CRISPR-U™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo. With CRISPR-U, Ubigene has successfully edit genes on more than 100 cell lines.
CRISPR/Cas9 recognizes the target sequence with gRNA, and guide Cas9 endonuclease to cut the upstream of PAM, resulting in the double-strand break (DSB) of the target site DNA. To repair the DSB, the cell uses its own DNA repair mechanism to add or delete or replace pieces of DNA sequences via Homology Directed Repair (HDR) or Non-Homologous End Joining (NHEJ).Close
CRISPR-U™ (based on CRISPR/Cas9 technology), developed by Ubigene, is more efficient than general CRISPR/Cas9 in double-strand breaking, and CRISPR-U™ can greatly improve the efficiency of homologous recombination, easily achieve
Gene-editing Cases : 100 Types of Cell Lines

Gene-editing Cases:100 Types of Cell LinesClick Here

Urinary System

Human bladder carcinoma cell line (TCCSUP)
Human bladder carcinoma cell line (5637)
Human Bladder Transitional Cell Carcinoma Cell Line (T24)
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)
Madin-Darby Canine Kidney Cell Line MDCK(NBL-2)
Human prostate carcinoma cell line (22RV1)

Blood and Lymphatic System

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
Rat Basophil Leukemia Cell Line (RBL-2H3)
Human leukemia cell line (HL-60)
Human T lymphocyte cell line (Jurkat, Clone E6-1)
Human myelogenous leukemia cell line (K-562)
Human caucasian histiocytic lymphoma cell line (U937)
Human B Cell Lymphoma Cancer Cell Line U2932
Human B Cell Lymphoma Cancer Cell Line OCI-LY3
Human Acute Non-B Non-T Lymphocytic Leukemia Cell Line (Reh)

Respiratory System

Human Bronchial Epithelial Cell Line (16HBE)
Human Lung Cancer Cell Line (A549)
Human Lung Cancer Cell Line (Calu-1)
Human Lung Cancer Cell Line (Epithelium)
Lewis lung carcinoma (LLC)
Human Non-small Cell Lung Carcinoma Cell Line (H1299)
Human Non-small Cell Lung Carcinoma Cell Line (HCC827)
Human Lung Adenocarcinoma Cell Line
Human Bronchial Epithelial Cell Line (BEAS-2B)
Human Bronchial Epithelial Cell Line
human embryonic lung fibroblasts
Human fetal lung fibroblast cell line (MRC-5)
Human hypopharyngeal carcinoma cell line (FaDu)
Human Lung Squamous Cell Carcinoma Cell Line NCI-H226
Human Small Cell Lung Cancer Cell Line H69AR
Chinese hamster lung cells (V79)

Endocrine System

Human Breast Cancer Cell Line (MDA-MB-231)
Rat Breast Cancer Cell Line (4T1)
Human Pancreatic Carcinoma Cell Line (PANC-1)
Human breast cancer cell line (MCF-7)
Human Breast Adenocarcinoma Cell Line (SK-BR-3)
Human Metastatic Pancreatic Adenocarcinoma Cell Line (AsPC-1)
Human Pancreatic Carcinoma Cell Line (MIA PaCa-2)
Human Pancreatic Carcinoma Tumor Cell Line
Mouse Pancreatic Carcinoma Cell Line (PANC-1)
Human Prostate Cancer Tumor Cell Line
Human prostate cancer cell line (PC3)
Human prostate cancer cell line (LNCaP)
Mouse Acinar Pancreatic Cell Line (266-6)
Human pancreatic cancer cell line (BxPC-3)
Human Breast Cancer Cell Line MDA-MB-468
Human Breast Cancer Cell Line MDA-MB-453
Human Breast Cancer Cell Line MDA-MB-436
Human Breast Cancer Cell Line ZR-75-1
Human Breast Cell Line MCF10A
Mouse Pancreatic Carcinoma Cell Line Capan-2
Human Ductal Carcinoma Cell Line BT-474
Human Prostate Cancer Cell Line VCaP

Circulatory System

Human Vulvar Leiomyosarcoma Cell Line
Rat Cardiac Myocytes (H9C2)
Human Coronary Artery Endothelial Cell line (HCAEC)
Mouse Myoblast Cell Line (C2C12)

Digestive System

Human Colon Cancer Cell Line (HCT116)
Human Colon Cancer Cell Line (SW480)
Human Colon Cancer Cell Line (SW620)
Human Colon Cancer Cell Line (HT-29)
Human Colon Cancer Cell Line (LoVo)
Human colon carcinoma cell line (RKO)
Human caucasian colon adenocarcinoma cell line (COLO 205)
Murine colorectal carcinoma cell line (CT26.WT)
Human colon adenocarcinoma cell line (DLD-1)
Human colorectal adenocarcinoma cell line (NCI-H716)
Human colorectal adenocarcinoma cell line (Caco-2)
Human colon carcinoma cell line (T84)
Human liver cancer cell line (Hep G2)
Human Hepatoma Cell Line (Hep3B)
Human hepatocellular carcinoma cell line (HuH-7)
Human Hepatocellular Carcinoma Cell Line
Rat Hepatoma Cell Line
Rat Hepatoma Cell Line
Human Primary Colon Cancer Cell Lines
Human Gastric Cancer Cell Line (HGC-27)
Human gastric cancer cell line (AGS)
Human Esophageal Squamous Carcinoma Cell Line
Human Esophageal Squamous Carcinoma Cell Line (KYSE-150)
Human renal cell carcinoma cell line (786-O)
Porcine small intestinal epithelial cell line
Murine Colorectal Carcinoma Cell Line MCA38
Human Hepatocellular Carcinoma Cell Line SNU-387
Human Gastric Cancer Cell Line SGC-7901
Human Esophageal Squamous Carcinoma Cell Line KYSE-150
Human Esophageal Squamous Carcinoma Cell Line KYSE-30
African green monkey kidney cell(Vero)

Skeleton, Articulus, Soft Tissue, Derma System

Human Osteosarcoma Cell Line (MG63)
Human bone osteosarcoma epithelial cell line (U-2 OS)
Human fibrosarcoma cell line (HT1080)
Human Osteosarcoma Cell Line
Immortalized Human Epidermal Cell Line
Mouse Chondrocyte Progenitor Cell Line
Ewing's Sarcoma Cell Line
Human Malignant Melanoma Cell Line
Human malignant melanoma cell line (A-375)
Murine melanoma cell line (B16-F10)
Human Melanoma Cell Line M14
Mouse myeloma cell line (Sp2/0-Ag14)

Ocular, Otolaryngologic and Oral System

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)

Brain and Nervous System

Human Neuroblastoma Cell Line (SK-N-SH)
Mouse neuroblastoma cell line (Neuro-2a)
Human Glioblastoma Cell Line (U251)
Rat Glioblastoma Cell Line (C6)
Human Glioblastoma Cell Line
Human glioblastoma cell line (U-87 MG)
Mouse Glioblastoma Cell Line GL261
Immortalize Human Microvascular Endothelial Cell Line hCMEC/D3
Mouse Hippocampal Neuron Cell Line HT22
Mouse Anterior Parietal Bone Cell Line (MC3T3-E1 Subclone 14)

Reproductive System

Human Cervical Carcinoma Cell Line (HeLa)
Human Ovarian Adenocarcinoma Cell Line
Mouse Embryonic Osteoblast Precursor Cells
Mouse Embryonic Fibroblasts (Preadipocytes)
Mouse Embryonic Fibroblasts (NIH/3T3)
Human Cervical Carcinoma Cell Line Hela 229
Human Cervical Squamous Cell Line SiHa
Human Ovarian Cancer Cell Line SK-OV-3
Human Ovarian Cancer Cell Line OVCAR3
Mouse Testicular Stromal Cell Line TM3
Chinese Hamster Ovary Cell Line (CHO-K1)

Knockout cell line
gRNA and Cas9 would be transferred into cells by virus transduction, liposome transfection or nucleofection. After drug screening, single clones would be generated. Positive clones would be validated by sequencing.。
Classification of gene knockout cell line
classification criteria Classification
Size of the Knockout Sequence Short fragment removal; frame shift mutation; Large fragment removal.)
Methods Virus transduction; liposome transfection; nucleofection.
Cell Types Tumor cells; other cell lines; IPS/ES etc.
Knockout Strategies
Short fragment removalShort fragment removal

Guide RNAs target introns at both sides of exon 2 and the number of bases in exon 2 is not a multiple of 3, which can cause frame-shift mutation.

Frame-shift mutationFrame-shift mutation

Guide RNA targets the exon, and the base number of deletion is not a multiple of 3. After knockout, frame-shift mutation would cause gene knockout.

Large fragment removalLarge fragment removal

Complete removal of the coding sequence to achieve gene knockout.

CRISPR-U KO Strategy diagram
Work Flow and Validation
Work Flow and Validation
Case Study
Chinese hamster ovary (CHO) cells have been used as host cells in the production of a range of recombinant therapeutic proteins, including monoclonal antibodies and Fc-fusion proteins. Host cell proteins (HCP) represent impurities that must be removed from therapeutic formulations because of their potential risks for immunogenicity. While the majority of HCP impurities are effectively removed in typical downstream purification processes, clearance of a small population of HCP remains challenging. Using the CRISPR/Cas9 system, Anxa2-, and Ctsd-knockout CHO cell lines were successfully established, and this study confirmed the complete elimination of the corresponding HCP in cell lysates. Importantly, all knockout cell lines showed similar growth and viability to those of the wild-type control during 8 days of cultivation. Thus, knockout of unrequired genes can reduce contamination with HCP in the production of recombinant therapeutic proteins.
(a)The constructed sgRNA targeted a unique sequence in exon 4 of the Anxa2 gene. The amplicon sequence was analyzed in both directions.
(a)The constructed sgRNA targeted a unique sequence in exon 4 of the Anxa2 gene. The amplicon sequence was analyzed in both directions.
(b)The constructed sgRNA targeted the unique sequence in exon 2 of the Ctsd gene.<br> The amplicon sequence was analyzed in both directions.
(b)The constructed sgRNA targeted the unique sequence in exon 2 of the Ctsd gene.The amplicon sequence was analyzed in both directions.
The constructed sgRNA targeted the unique sequence in exon 2 of the Ctsd gene.The amplicon sequence was analyzed in both directions.
Characterization of protein expression from CHO knockout cell lines by SDS-PAGE and western blotting analysis.
(a) Anxa2-knockout cell lines.
(b) Ctsd-knockout cell lines. Cell culture supernatants and cell lysates were subjected to SDS-PAGE. Total proteins were detected by CBB staining. Western blotting analysis of each protein was performed using respective capture and detection antibodies.
The asterisk indicates the nonspecific band. The double asterisk indicates what is likely a fragment of cathepsin D.
Annexin A2 and cathepsin D were not detected in the cell culture supernatants or lysates of the corresponding knockout cell lines, suggesting successful exclusion of these impurities from host cells for therapeutic protein production. In addition, no truncated HCP was observed in the protein expression analysis of the Anxa2 and Ctsd knockout cell lines.
Reference:
Fukuda, N., Senga, Y., & Honda, S. (2019). Anxa2‐and Ctsd‐knockout CHO cell lines to diminish the risk of contamination with host cell proteins. Biotechnology progress, e2820.