Knockin Cell Lines

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Knockin Cell Lines

·    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).

Check out our list of cell lines that we had successfully modified
·    Technical advantages    ·

Exclusive innovation, 10 times more efficient in gene-editing.

Successfully edit genes on more than 100 types of cell lines.

Easily generate knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo.

CRISPR-U™ offers a 100% mutation guarantee. No mutation, no charge!

·  Knockin Cell Line  ·
The cells would be co-transfected with gRNA, Cas9 and donor vector by electroporation, and then screened. After drug screening, single clones would be generated. Positive clones would be validated by sequencing. Cells knockin with reporter genes can also be selected by observing the fluorescence protein.
·  Knockin Strategies  ·

· Protein fusion

Guide RNA and Cas9 complex cause a double-strand break (DSB) on the target site of DNA. The donor vector carrying knockin sequence is the template for homologous recombination repair (HDR), and it recombines to the target site.

· Replacement of specific locus:

·    Work Flow and Validation    ·
·    Case Study  ·
T cells carrying chimeric antigen receptors (CARs) can mediate tumor rejection, which is an effective way to cure B cell malignant tumors. By using CRISPR/Cas9 technology, TRAC gene was replaced by CD19 specific CAR sequence.CAR expression in human peripheral blood T cells was found, and the immune response ability of T cells was enhanced.
a

CRISPR/Cas9 mediated CAR gene integrated into TARC site. 1928z is a CD19 specific CAR sequence.

b

TCR/CAR flow plots.4 days after co-transfection of Cas9, gRNA and donor vector, CAR proteins were detected.

Reference:
Eyquem, J., Mansilla-Soto, J., Giavridis, T., van der Stegen, S. J., Hamieh, M., Cunanan, K. M., ... & Sadelain, M. (2017). Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature, 543(7643), 113.

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