How to perform a CRISPR knockout experiment?

CRISPR/Cas9 is an essential tool for generating genetic models for preclinical research, developing new treatments for genetic diseases, and modifying the genome of plants and animals. 

Here are the steps to perform a CRISPR knock out an experiment:

1. When designing a knockout experiment, you should gather all the information available about your target gene and cell line. This information will determine the complexity of the investigation. You must understand:

- cell ploidy

- sequence of the target gene

- possible phenotype linked to the knockout

2. Design single-guide RNAs with the least possibility of off-target effects. In this step, you should consider splice variants and check for microhomology. It is also good to check if there are no SNPs in your target region, which can be done by using PCR. You can choose a predesigned sgRNA as well. 

3. Choose the best CRISPR/Cas9 delivery method for you. It can be electroporation, AAV virus, or RNPs. 

You can check knockout efficiency using a particular assay. 

4. Characterize the clonal cell lines to ensure you silenced the target gene, and no off-target effect is observed. 

The techniques involved are indel identification, RT-PCR, Western blot, and karyotyping. 

Source:

https://www.takarabio.com/about/bioview-blog/gene-editing/successful-knockout-experiments-part-ii