You have sweated at the lab bench and in the cell culture suite for weeks (or more likely months) to plan and optimize for successful genome editing. Finally, you’ve got the guide RNAs and the vectors into the cells. Yes! But your work is not yet finished.
You can’t take for granted the fact that you’ll achieve CRISPR success, because, despite the explosion in its application in genetics labs around the world, genome editing is well known to be a bit of a dark art.
But don’t worry. Below is an exhaustive list of the steps that you can take to perform CRISPR validation.
Check the Deletion
If a straightforward deletion was your intention then the first step towards confirmation of your (hopefully) CRISPR success is to use a bit of basic PCR, an agarose gel, and some simple mathematics. To verify that the region you expected was deleted, you should:
- Design primers that will amplify the region around your editing. The amplification primers should encompass the sequence to be deleted and should extend at least 100 base pairs (ideally somewhere around 200-400 base pairs) on either side of the deletion. The amplicon (PCR product) should be large enough that you can easily see it on an agarose gel, but small enough that the deletion will cause an obvious shift in the size of the band on the gel.
- Keep a note of the size of the amplicon and the size of your expected deletion.
- Extract DNA from both your CRISPR-Cas9 genome-edited cell population and your unedited control population.
- Perform the PCR using the extracted DNA from both the edited and unedited cell populations.
- Run the PCR products on an agarose gel for a length of time sufficient to allow separation of the edited and unedited bands.
- Confirm that the expected size of deletion corresponds to the reduction in the size of the PCR product.
- Proceed to the next step of the verification process!
In cases where a more sensitive method is needed, you can validate your CRISPR using TIDE (Tracking of Indels by Decomposition) assays.
Sequence Your PCR Products
The next step of the verification process is to use Sanger sequencing to verify that the intended region has been deleted. However, this step can be made slightly tricky by the fact that the CRISPR-Cas9 system rarely modifies the DNA at exactly the nucleotide expected. Often, it will cut the DNA up to three base pairs away from the excision site. This is problematic for Sanger sequencing because it can create a mixture of products that vary by one, two, or three nucleotides in length.
In practice, this means that the chromatogram before the deletion site can be read easily, but after the deletion site, the sequencing trace will give scrambled results. The workaround is to sequence the PCR product in both the forward and reverse directions, in order to map both upstream and downstream of the deletion.
Measure Gene Expression
If the region you’ve edited is non-coding then it’s likely you wish to determine if it is involved in regulating gene expression. To determine this, a simple qRT-PCR experiment will enlighten you. Extract RNA from the cell populations both with and without the deletion. Synthesize your cDNA, quantify mRNA expression levels then compare between the two groups.
Measure Protein Expression
If you’re aiming to knockout a gene, then the mRNA expression levels may not necessarily be altered by your genome editing. However, the protein almost certainly should be. The best way to determine this is to perform a western blot. This requires extraction of protein from your cell population carrying the edited region, as well as from the control cell population.
The western blot should be performed with a primary antibody that recognizes an epitope towards the C-terminus of the expected deletion. An antibody that recognizes an epitope towards the N-terminus of the deletion will not give you the information that you need as this part of the protein may still be translated. Cross your fingers and hope that you see an appropriately-sized band in the control lane, and no band in the deletion lane! If this step confirms that you have successfully deleted your protein, then proceed to the next verification step.
Measure the Impact in Your Cells or Model System
Once you have deleted your genomic region and confirmed that the protein has been deleted, then the fun part begins! Now you can move on to investigate the resultant phenotype of your cells or model system which no longer expresses your gene or protein of interest.
Be sure that any experiment that you perform with the cells carrying the deletion, you also perform that experiment using the control cells under the exact same conditions.
Share Your CRISPR Success with Anyone and Everyone!
Genome editing is a long, complicated and difficult process. It takes skill to successfully edit a genome, so if you are able to do so then you are perfectly entitled to share that CRISPR success with your supervisor, colleagues, and even your friends and family! It will also provide an exciting and pivotal section of your thesis or next publication, providing novel data and insight into biological processes. But more than that, it proves to the world that you are a talented and able scientist.
Discover more about CRISPR in the Bitesize Bio CRISPR Research Hub.