A new application note using BMG microplate readers recently went online from entitled: CRISPR/Cas 9 genome-edited cells express nanoBRET-donor that monitors protein interaction and trafficking. Genome editing has reached a new level with the application of CRISPR based technology. It is not only extremely useful for use in the lab, as is reported in the application note, but also it touted for its potential as a therapy for genetic diseases.
The new application note features the work of the lab of Kevin Pfleger. Kevin has done some really outstanding work recently down in Australia and has been good enough to contribute to application notes, a webinar and a SLAS tutorial with BMG in recent years! The new note is really excellent and you should check it out.
But we first want to focus on the genome editing (or sometimes simply gene editing) that is used in the note. Although a variety of different approaches have been used to perform genome editing they have almost universally been replaced by the CRISPR/Cas9 approach.
So what is CRISPR/Cas9? Lets’ start with what that acronym stands for: Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9. The name CRISPR was applied before their function of these sequences was known (not to mention its potential). So I guess we will have to forgive the scientific community for coming up with a name that is also part of your refrigerator. It does accurately describe what these sequences look like in the prokaryotes where they were first seen.
The CRISPR/Cas system was characterized for its role in a prokaryotic immune system where it confers resistance to foreign (especially viral) genetic elements. The repeating code in the bacteria’s genetic sequence is interspersed with “spacer” sequences that are actually pieces of genetic code from previous invaders. If the same invader returns these sequences, when transcribed into RNA, are able to guide the Cas enzyme to bind the invasive DNA, cut it up and shut down the gene. The CRISPR system is found in around 40% of all bacterial species and in 8 out of 10 archaea.
What is remarkable about this system is the specificity with which it can target a DNA sequence. It is this specificity that was noticed and which has been subsequently exploited in its’ role as a genome editor. In human and animal applications the RNA that seeks out viral DNA is replaced with an RNA sequence that will target a specific gene. In this way you can see how this approach could be used to silence a particular gene once the CRISPR/Cas9 interacts with nuclear DNA. The break in the DNA will be repaired, but the problem with this most basic approach is that the most common repair mechanisms can result in diverse mutations (deletions, insertions, etc.) that can lead to premature stop codons. Therefore validation of the particular effects will be needed before you proceed with experiments.
One of the main differences in using this system in the lab is that the cells involved are usually eukaryotes. So the DNA that needs to be accessed is in the nucleus. Typical transfection techniques work to get the CRISPR/Cas9 system expressed in a particular cell and you could wait for the cells to divide, when the nuclear envelope is temporarily absent. But that is not usually suitable. One approach employed is to deliver the CRISPR/Cas9 system to the cell and into the nucleus with a nanoparticle.
Of course CRISPR/Cas9 has not only been used in gene silencing. In the case of the recent BMG app note mentioned previously they modify endogenous CXCR4, a G-protein coupled receptor, so that it expresses a tag, NanoLuc (NLuc). How is this done? Basically once you have the DNA cut at the desired location a donor DNA template will allow you to insert the desired sequence into the place where the DNA is cut. Homology-directed repair will complete the insertion of the donor sequence. Not at all a simple task, but it has been done successfully and there are groups like AddGene that provide information and resources to help.
The benefits of adding a tag through gene editing are that the tagged-protein of interest is expressed at endogenous levels. It has always been a concern that the conclusions drawn from exogenous expression (usually overexpression) are artefactual. This approach will decrease that concern. Furthermore the protein expression should be regulated in an appropriate manner so the door is open to investigate if a proteins partner or movement within the cell changes during different developmental stages, for example.
In the application note, the NLuc-tagged CXCR4 protein was used in a number of different BRET assays to look at association of the receptor with different component under a variety of conditions. One approach of particular interest was to look at receptor internalization and trafficking. This was achieved by taking the cells that already expressed CXCR4-NLuc and transfecting them with K-Ras with a HaloTag and Rab4 tagged with Venus fluorescent protein. K-Ras is well characterized to be localized to the cell membrane Rab4 is an early endosome marker. Venus and Halo Tag emit at different wavelengths of light but can both be excited by the light produced by NLuc. The CLARIOstar LVF Monochromator could be set up to monitor Venus (emission wavelength-band pass = 550-60) and HaloTag (660-100).
As for CRISPR as therapy, CRISPR therapeutics is seeking permission to test their drugs on humans to treat the blood disorders beta thalassemia and sickle-cell. The potential for treatment of genetic disorders using CRISPR has been tantalizing ever since the technology came on the scene and there 3 companies currently working toward CRISPR based therapeutics. Are we ready? Putting aside any philosophical debate about whether genome editing it is ‘right’, there appear to be a number of hurdles that must be overcome before CRISPR therapies achieve widespread use. Of course this is still true of any traditional drug. So as with traditional drugs the different CRISPR therapies will by necessity need to be evaluated for potential side effects on a case by case basis. But considering it has only been 5 years since the first modifications of mammalian cells was performed, the technology has come a long way! With continued efforts to characterize better methods of delivery and potential side effect of this technology proceeding at a rapid pace we could see real therapies very soon.