Ubigene Biosciences: Point mutation cell line generation--Ubigene Weekly Q&A

Nowadays, many researches indicate that CRISPR/Cas9 is becoming the optimal tool to construct the models of human diseases, such as human pluripotent stem cells and tumor cell lines. With modern sequencing, CRISPR/Cas9 can introduce precise point mutations (homozygous or heterozygous) in various cell lines and enables to construct human disease models. This is highly advantageous for studying both the therapy and the underlying genome of the diseases.

We also received a few questions this week about introduction of point mutation in cells using CRISPR/Cas9. Below are the Q&As for this week.

Q1 How can I use CRISPR/Cas9 technology to study gene point mutation cell lines?

The specific mutations can be achieved by CRISPR/Cas9 efficient introduction of targeted double-strand breaks (DSBs), repaired using DNA repair donor (e.g., single-stranded oligo DNA nucleotide, ssODN) under homology-directed repair (HDR) system. Importantly, with the KI cassette based on the CRISPR/Cas9 system, we can also selectively introduce mono-allelic and dual-allelic sequence changes, which are heterozygous and homozygous point mutations to construct pathogenic disease models.

Homozygous point mutations require gRNAs to target positions close to the target mutation site, whereas heterozygous point mutations can be achieved by distance-dependent incorporation of suboptimal mutations or by using mixed repair templates.

So, the introduction of point mutations in cell lines mediated by CRISPR/Cas9 technology is very useful for the studies on the gain of function or loss of function.

Q2 Hi there, I want to construct a gene point mutated Hep-G2 cell line. For reference, could you tell me the estimated timeline for the whole experiment?

For endogenous point mutation, it’s a bit challenging to introduce in Hep-G2 because the proliferation rate for Hep-G2 is relatively slow and the single-cell colony formation rate is relatively lower than other common cell line models. If everything goes smoothly, the whole process takes about 3-4 months. Because it is difficult and time consuming, you could consider outsourcing the experiment.

Ubigene is experienced in successfully modifying Hep-G2 cell line and if you order point mutation cell line generation services from Ubigene, we will give you the KO cell line for free, making your experiment and researches more convenient.

Q3 Hi there, I want to introduce a point mutation (change of an amino acid) in the target gene using CRISPR/Cas9 technology, and I want to do cell transfection and single-cell clone isolation in my own lab. I have searched many articles and checked some information from addgene, found that I need to construct a donor DNA to achieve the point mutation in the cells. How to design the constructs and how much budget is generally required?

The donor DNA is not as complicated as you think to construct. A donor comprises a sequence carrying the mutation and upstream and downstream sequences (each about 1-2Kb long) for the purpose of homologous recombination. Or you could also use oligo which has shorter upstream and downstream sequences (about 100-200bp) to achieve the point mutation.

For the question of budget, the regular price for a set of point mutation constructs and oligo is about 770 USD in Ubigene.

Above are the Q&As of the week.

Feel free to contact us if you have any similar questions.

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