The gRNA vector (containing Cas9) of the CRISPR/Cas9 system and the Donor carrying target mutation
or target
gene sequences will be co-transfected into cells by electroporation. Then the gRNA and Cas9 complex
will cause a
double-strand break (DSB) on the target site of DNA and the cells will undergo homologous
recombination repair
(HDR) using Donor as a template to recombine target mutations to the target sites. After antibiotic
screening,
single-cell clones will be generated. Positive clones that are successfully introduced the target
mutations be
validated by target site amplification and sequencing. Final deliverables will be the positive cell
clones,
related data and project reports.
Knockin Cell Service Details
Cell type
Various types of cells including tumor cell lines, regular cell lines,
IPS/ES cell lines
Human Breast Cancer Cell Line(MCF7)Mouse insulinoma β cell line(NIT-1)Human Breast Cancer Cell Line(JIMT-1)Human breast cancer cell line(T-47D)Human pancreatic cancer cell line(BxPC-3)Mouse Acinar Pancreatic Cell Line(266-6)Human Prostate Cancer Cell Line(VCaP)Human Pancreatic Carcinoma Cell Line(MIA PaCa-2)Mouse medullary breast cancer cell line(E0771)Mouse pancreatic cancer cell line(Pan02)Human Metastatic Pancreatic Adenocarcinoma Cell Line(AsPC-1)Human Breast Adenocarcinoma Cell Line(SK-BR-3)Human Pancreatic Carcinoma Cell Line(PANC-1)Rat Breast Cancer Cell Line(4T1)Human Breast Cancer Cell Line(ZR-75-1)Human Breast Cancer Cell Line(MDA-MB-231)
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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