CRISPR stands for Clusters of Regularly Interspaced Short Palindromic Repeats. Dr. Jennifer Doudna was one of the first researchers to see these short palindromic repeats in bacteria and archaea where she speculated that they were being used as a form of molecular immune system to protect these organisms from viruses.
Even bacteria get sick, so having a protection against invading viruses is a matter of life and death to a cell. Recall that viruses are essentially genetic material that will reproduce itself again and again after it hijacks a cell. Viruses may have protein coats or membranes to protect them outside of the cell, but inside, they are little more than DNA. If this DNA can be damaged or destroyed, then the virus is rendered harmless.
To the right is a clip from Dr. Doudna’s video illustrating the repeated elements (in black) flanking a variety of ‘other DNA’. This ‘other DNA’ is what the cell will use to identify foreign DNA – presumably from retaining the genomic material from an earlier exposure either in the lifetime of the single cell or its parents.
So, how does it actually work?
Two videos do an excellent job of explaining how CRISPR works. A short, simple video from MIT gives a non-technical explanation (a good place to start).
MIT’s McGovern Institute
Jennifer Doudna explains the system in greater detail…
Basically, the natural system uses two RNA molecules to target specific DNA sequences in the genome and recruit a protein that acts as an endonuclease to cleave this target:
crRNA – a ‘targeting’ molecule
tracrRNA – an adaptor RNA that recruits CAS9 to the bound crRNA
CAS9 – an endonuclease enzyme that will bind and cleave DNA once recruited by the RNAs
Doudna’s lab improved the system by combining the two RNA molecules into a single RNA that still effectively recruits CAS9 but is easier for researchers to manipulate in the lab. This last element is essential because manipulating this RNA sequence gives researchers the power to target any DNA sequence in the cell.
As stated above, the system was originally identified in prokaryotic organisms where it appears to allow targeting of the viruses that attack them. CRISPR uses ‘stored’ DNA as the targeting RNA and then brings in CAS9. CAS9 binds to the targeted DNA and cleaves it resulting in one of two possibly outcomes. 1) the virus is destroyed and is no longer a problem, 2) the virus is cut, but then repairs itself – hopefully in a way that introduced fatal mutations.
How might this translate into clinical medicine?
The possibilities are endless, however a few low-hanging fruit present themselves immediately. Among these are therapies for sickle cell anemia (and a host of other blood disorders). Because sickle cell anemia is caused by a single base pair mutation, it is conceivable that hematopoietic (i.e. blood) stem cells can be isolated, the faulty gene repaired, and then re-introduce the corrected stem cell back into the body (possibly after the faulty stem cells have been ablated).
The newly altered and re-introduced stem cells now do the rest of the work for you by finding their place in the body where they reside while continually producing cells with the desired genetic changes.
The key is that these RNA molecules are quite simple to make exactly and in pure form (i.e. they can be manufactured chemically rather than needing cells to do the job for us and then we have to clean up all the extraneous contaminants). Most labs will design the molecules in-house and then order the constructed molecules from a ‘core lab’ that specializes in doing just that.
Jacob Corn, of UC Berkeley has compiled a simple protocol that anyone with a modicum of molecular biology training could follow. Find that protocol here.