How to make good use of CRISPR/Cas9 technology when constructing knockout cell lines?

From January 2013 to the present, in the past 2 years, the application field of CRISPR/Cas9 technology has been continuously expanded by the world's outstanding scientists. These include gene knockout, site-directed mutagenesis, site-directed integration, gene silencing, gene mapping and CHIP, and the application of this technology is constantly expanding.

Gene knockout using CRISPR/Cas9 technology is the most efficient and cost-effective technique compared to other recombinant technologies. The key factors are the same as the following:

1) Identify the gene sequence that needs to be knocked out

First, you need to identify the intron and exon sequences; you can log in to the NCBI or UCSC website to find it easily;

2) Suitable target site selection

It is generally recommended to design 2-3, then transfect into cells, use CruiserTM and sequencing to verify which target site is the best; or log in to the CRISPR/Cas9 knockout vector library: ( see the vector library detail page ) See if you have the genes you want to study. The target sites in this vector library are verified by activity in the cells to ensure knock-out activity;

3) Knock out carrier selection

An empty vector, a resistant vector (Puro/Neo), a fluorescently labeled vector (EGFP/RFP), a double knockout vector, and the like can be selected as needed. Among them, the empty vector is small (8K), which can improve the efficiency of transfection; the resistance is convenient for subsequent resistance screening, and the fluorescent labeled carrier is convenient for flow sorting;

4) Single cell growth

The growth of individual cells in different cells is different. Some cells grow fast, some cells grow slowly, and some single cells are almost not long. This requires a different approach. Here is a small single-cell culture consumable: Cell PlazaTM. The design is exquisite, using the principle of co-cultivation. If you encounter a single cell that is not long, you can try it. At the same time, this is very helpful for the growth of small callus when plant cells are knocked out. Researchers working on plant gene knockouts can also try it out;

  5) Positive clone screening

In addition to knocking, CRISPR/Cas9 can also be applied in all directions, such as site-directed mutagenesis, site-directed integration, gene silencing, and gene mapping, with different details in each direction. I will not explain them here. If you need more information, you can contact us at any time. You can also pay attention to the official WeChat (WeChat: Genloci) of Jirui Bio, and participate in the interaction.