Analyzing Your First CRISPR Experiment

If you are reading this, by now you are more than familiar with the concept of genome editing and CRISPR/Cas. Over the last weeks, we discussed the main elements to factor in when designing a CRISPR/Cas sgRNA approach and introduced CHOPCHOP as a helpful tool to help up in this regard.

Today we will discuss how we can assess whether CRISPR/Cas did its job: to cut the DNA.

A little warning before we start, this is gonna be a longer article than usual. This is because we need to explain some concepts in more detail, but don’t worry, it’s going to be worth it and, more importantly, it’s going to be fun!

Using PCR to assess gene editing efficiency

I know, you’re thinking “Hold on, my friend! How did the Cas9 and the sgRNA get into our cells in the first place?”

Glad that you are asking, and we did not forget about it. You see, CRISPR/Cas is delivered to cells in the same way other transgenes, nucleic acid or recombinant proteins are delivered. Meaning using either chemical or electrophysical methods to help the cargo pass the cell membrane.

Even so, “Delivery” is not a trivial issue, and we will discuss it in the near future. For now, suffice it to say that after a smart design of your sgRNA – you can thank us later - and a textbook transfection, we are finally at the point where we want to know whether we managed to CRISPR some cells.

Let’s take the Genetics101 book and go to the page describing that a genetic trait possesses a Genotype – the genetic sequence that codifies it – and a Phenotype – the result in the “observable world” of that genetic sequence -.

As such, the result of a gene editing experiment can be assessed in two ways. We can look at the genetic level -did anything change in the DNA sequence?- or, in case the target sequence codifies for an mRNA or -even better – a protein, we can also look at the phenotype.

Let’s start with the DNA, as this will be the most common and trustworthy approach.

How do we do that? Once a professor told me “In case of doubt, most molecular biology questions can be answered with a Polymerase Chain Reaction (PCR)”.

And this is the case!

Whether you are aiming for basic Sanger sequencing-based approaches or are fancying the latest in Next-Generation Sequencing, make peace with the fact you will need to face the dreadful PCR.

Routinely, we may use three different methods, characterized by an increasing level of sensibility, but they all start from a PCR: Polymorphism-like-Analysis, Sanger Sequencing and NGS-based.

The first step is thus to extract the DNA from the treated cells and amplify via PCR the target sequence. Usually, this procedure entails amplification of an area with a length between 300-nt ( max length for NGS) and 500 nt (recommended length for Polymorphism-like-Analysis and Sanger sequencing) around our target sequence, so that we can study any changes to that region caused by our CRISPR experiment. Very importantly, for all essays, we will want a PCR product coming from the DNA of CRISPR-treated cells, as well as a PCR product from untreated cells, which we will use as a negative control and as a reference sample for assays relying on DNA sequencing.

Now that we have our target sequence amplified as a PCR product, also known as amplicon, it is time to run your assay. We just need to choose which one.

Polymorphism-like analysis

This kind of assay takes advantage of a class of restriction enzymes that can identify and cut bulges present in the DNA structure when two bases in the complementary strands are not complementary and cannot pair with each other. To do this assay, the amplicons of the PCR reaction are heated up to 95-98ºC for a few minutes, which causes the two strands to separate (denature). Then, the amplicon is cooled down to room temperature, which will cause the separate strands to couple again. Interestingly, the strands that were separated are unable to find their original partner and will bind to each other randomly. Since the PCR amplicon comes from the DNA of the different cells in our sample, which will contain a mix of edited and unedited cells, there’s going to be variability in the sequence of our PCR amplicons. Even those DNA molecules with edits may also be different from each other, because the mutations resulting from NHEJ repair are not homogenous, as we described earlier.

However, this is not the only way we can do this. For example, if a protein is expressed on the surface of the cell, like a receptor, then it may be possible to use Flow Cytometry. In this kind of experiment, we will still use a labeled antibody, but in this case, the signal would be detected not via Western blot but by detecting and quantifying the presence of labeled r cells using a laser-based system. This system will recognize the cells where a labeled (fluorescent) antibody has attached to a surface receptor and can even allow us to separate labeled and unlabeled cells for later experiments. Compared to Western Blot, Flow Cytometry is more quantitative, easier, and faster to perform. However, it can be mainly applied to surface receptors and is not useful for proteins that don’t stick out on the outside of our cells. While there are cell permeabilization techniques that allow us to look at the inner content of the cells, these techniques are more complex and may require some optimization. Sometimes, a Western Blot is actually the best option, unfortunately!