Top 4 Reasons: Why Use CRISPR-Cas9 Antibodies and How?

Fri, 12/09/2016 - 14:37

1. Verification of the success of transfection

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).    

Chromatin immunoprecipitation (ChIP) with CRISPR-Cas9 antibody.

CRISPR-Cas9 Antibody (6G12) - C-Terminus [NBP2-52398] 
NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9 (flag tagged), and a GFP-targeting gRNA were fixed with formaldehyde, harvested and sonicated to get 200-500bp DNA fragments. 50ug chromatin was incubated over night at 4C with the indicated antibodies (200ul hybridoma supernatants of clones 7A9 and 6G12; 5ug Flag antibody) followed by incubation with protein G beads for 3h at 4C. After washing chromatin was eluted from the beads and crosslinking was reversed over night at 65C. After a proteinase K digestion step, DNA was separated using phenol/chloroform/isoamyl alcohol (PCI), precipitated with ethanol/sodium acetate and dissolved in water. For qPCR, primers either targeting the GFP gene or as negative control non-targeted regions (Ppap2c +7122 and Prkcd +24069 from transcription start) were used.

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


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