Point Mutation

Homepage > Gene Editing Services > Stable Cell Lines >

Point MutationService

·  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
·  Point Mutation Cell Line  ·
The cells would be co-transfected with gRNA, Cas9 and donor oligo by electroporation, and then screened. After drug screening, single clones would be generated. Positive clones would be validated by sequencing.

Point mutation Strategy:

·    Work Flow and Validation    ·
·    Case Study    ·
The A79V mutation of PSEN1 gene can cause Alzheimer's disease. Somatic cells of patients with Alzheimer's disease are induced into pluripotent stem cells (iPSCs), and then the mutant gene is modified by replacing the point mutation with a wild-type sequence. By studying the iPSC of patients and the modified iPSC, we can know the effect of the mutation on cell phenotype, so as to further study the pathological effect of the mutation.
CRISPR/Cas9 and ssODN used to repair the point mutation in A79V-hiPSC. A) Genomic sequence surrounding the mutation site: mutated nucleotide (T, red); sgRNA recognition site containing 20 bp (yellow); CRISPR cutting site between the 17th and 18th bp (bold); forward and reverse primers (pink). B) ssODN with 120 bp, 60 bp upstream and 60 bp downstream the mutation site containing the WT nucleotide (C, green).
B. Sequencing of exon 4 of the PSEN1 gene in hiPSCs.

A) Heterozygous c.236C>T substitution in the mother line previously published.
B) Successful correction of the point mutation (T>C).

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.

Recommended products


×

Search documentation

Literature:

Name: *

Company: *

Telephone: *

Email:

Notes: After submitting the order, we will contact you as soon as possible.

request now