Why- If the CRISPR-Cas9 transfection is not successful, it would not be relevant to relate the observations from transfected cells to the expected outcome of gene editing experiment.
How- CRISPR-Cas9’s successful transfection can be verified through its detection at the protein level by employing Western blot or confocal staining analysis of cells that were subjected to CRISPR-Cas9 transfection.
2. Localization of CRISPR-Cas9 at subcellular level
Why- CRISPR-Cas9 must translocate to the nuclei of the transfected cells for executing its nuclease activity on the genomic DNA. Therefore, one must check that the Cas9 protein is actually being delivered into the nucleus.
How - The sub-cellular localization of Cas9 may be checked through ICC/IF or IHC staining of the transfected cells with CRISPR-Cas9 antibodies using nuclear staining protocol (with adequate cell permeabilization).
3. Confirming the target binding of CRISPR-Cas9
Why – CRISPR-Cas9 genome editing system has two major components: a synthetic RNA “guide RNA” (gRNA) and a non-specific CRISPR-Cas9 protein with nuclease activity. gRNA has a “scaffold” sequence which facilitates Cas9-gRNA binding, and a user-designated “spacer” or “targeting” sequence corresponding to the genomic target. Therefore, one must check if CRISPR-Cas9 is actually binding to the correct region on the DNA.
How- Using ChIP grade CRISPR-Cas9 antibodies (such as NBP2-52398), it is very easy to detect/quantify the binding specificity of Cas9 enzyme with a given gRNA and targeted or non-targeted region primers (see the data image shown below as an example).
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CRISPR-Cas9 Antibody (6G12) - C-Terminus [NBP2-52398] |
4. Quantification of the CRISPR-Cas9 expression levels
Why- The level and/or the duration of expression of Cas9 enzyme is very critical when using CRISPR-Cas9 genome editing technology. An extremely high expression level of Cas9 in stable clones can lead to non-specific activity. Similarly, in transient transfections, a chronic or extended expression of Cas9 protein can result in the generation of more off-target mutations.
How – In stable clones, it is suggested to isolate multiple clones and to screen them for Cas9 expression levels through Western blot analysis. On the other hand, the transitional nature of Cas9 expression in transient transfectants may be checked through Western blot analysis of the transfected cells collected at various time points.
Also see: Frequently asked questions (FAQs) on CRISPR-Cas9 genome editing
