Generating knock-out cells using CRISPR-Cas9
Based on the CRISPR-Cas molecular mechanism, the CRISPR-Cas technique can be used to generate knock-out cells by using the correct CRISPR-Cas components: the Cas9 endonuclease as the Cas protein and a gRNA sequence that targets the gene to be edited.
Once the CRISPR experiment has been performed, there are four main steps to follow in order to confirm that the selected gene was correctly knocked-out:
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Select the positive clones and make stable cell lines of them: This step relies on the addition of antibiotics to the cell culture. The plasmid inserted into the cells contains a fragment that confers resistance to a specific antibiotic. By adding the antibiotic over several days, only cells containing the plasmid and, therefore, resistant to the antibiotic will grow. It is a common procedure to select different clones for further analysis.
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Amplify the region of interest from the selected clones: Once the stable clones have been selected, a PCR is performed to amplify the target DNA region. It is important to perform the PCR on genomic DNA, as if the gene has been successfully knocked-out, it will not be expressed. Thus, RNA extraction and subsequent RT-PCR is not recommended for this step.
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Sequence the amplified region: In order to ensure that the DNA sequence has changed, the amplified DNA should be sequenced to evaluate what type of mutation has been inserted during the NHEJ process.
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Repeat detection method to observe if there are changes in the expression of the selected gene as well as in the cells' phenotype: This can be achieved using immunofluorescence, cell sorting, or western blotting.
If the cells were knocked-out correctly, the functions of the selected gene should be altered or completely disabled.