Although invaluable, GFP and its brethren are no longer particularly novel tools, whereas the CRISPR/Cas9 genome-engineering tool is revolutionising molecular biology as I write. CRISPR is a somewhat daunting acronym and “genome-engineering” sounds like something from a novel about the dystopian future. Let me explain (bear in mind that I spent my summer at Cold Spring Harbor Laboratory using and further developing the CRISPR system so I might be slightly biased).
The term CRISPR, first coined in 2002 (Jansen et al. in Molecular Microbiology), stands for Clustered Regularly Interspaced Short Palindromic Repeats; these refer to repeated sequences of DNA found in bacterial and archaeal genomes, which “spell out” the same sequence when “read” from either direction (i.e. they are palindromic). Cas9 is short for CRISPR-associated; Cas9 refers to an enzyme, which cuts double-stranded DNA (i.e. it is a nuclease). Unlike GFP, whose original function is still unknown, the purpose and to a large extent the mechanism by which CRISPR/Cas9 acts has been elucidated. Until recently it was believed that only vertebrates can mount an adaptive immune response. However, research into CRISPR/Cas9 has revealed that bacteria and archaea (the two domains of prokaryotic life) use this system as a rudimentary, hereditary adaptive immune system, albeit mechanistically very different from our adaptive immune system.
Whereas we try to stave off bacterial, viral or parasitic infections, these single-celled organisms try to protect themselves from invading bacteriophages (viruses that infect bacteria) or plasmids (semi-autonomous pieces of DNA). In 2007 (Barrangou et al. in Science) the first paper was published, indicating that the CRISPR DNA arrays may be involved in immunity. The experiments were done on the bacterium Streptococcus thermophilus: when CRISPR DNA sequences corresponding to bacteriophage DNA were deleted in previously resistant bacteria this led to the bacteria becoming susceptible to invading bacteriophages. And vice versa when novel DNA sequences, corresponding to bacteriophage DNA from a bacteriophage that had not been previously encountered, were inserted into the bacterial genomes this created new resistances. This is the simplified mechanism: bacteria/archaea incorporate some of the invading agent’s DNA into their own genome and upon re-infection they transcribe these DNA sequences into RNAs, which in turn associate with the Cas9 nuclease. The Cas9 nuclease is then guided to the DNA sequences in the bacteriophage or plasmid that are complementary to the guide RNA and can subsequently cleave this DNA, thereby rendering it non-functional.
So how does this simple yet ingenious immune system tie in with genome-engineering? Well, imagine that the RNA that guides Cas9 to DNA can be designed so that the nuclease no longer cuts plasmid or bacteriophage DNA, but any sequence of DNA that you happen to be interested in. This certainly does not mean that CRISPR/Cas9 will be used to “engineer” human genomes into alien genomes. What CRISPR can do is to manipulate the DNA of cells in culture or of animal models – mainly this has involved trying to knock-out the function of a particular, targeted gene, or to restore the function of a defective gene by providing a wild-type DNA template. First and foremost, therefore, the CRISPR/Cas9 technology is being used as a molecular biology tool that has made it far easier to manipulate DNA in an experimental setting than ever before. It’s so simple, in fact, that I could learn how to use it during the summer.
However, as with all great technologies and inventions the CRISPR system has some disadvantages that need to be addressed. For example, in its original application, cutting DNA with the Cas9 nuclease is an irreversible step, but in some experiments it can be beneficial to be able to switch between the two states. Furthermore, and more problematically, there are still a lot of improvements to be made with regards to the design of the guide RNA, which brings the Cas9 to the target DNA. Although there will probably always be some off-target effects (i.e. Cas9 cutting at locations that were not intentionally targeted because the guide RNA usually binds weakly to other DNA sequences), more and more programmes are being developed to try to minimise these effects.
Apart from in the lab, will CRISPR/Cas9 have applications therapeutically? Yes, hopefully, but probably not in the next couple of years. Earlier this year, for example, it was shown in mice that CRISPR/Cas9 delivery can correct mutations that lead to a disease similar to human hereditary tyrosinaemia, a rare liver disease (Yin et al. in Nature Biotechnology). Gene therapy using CRISPR is certainly imaginable, but will need to be thoroughly tested before application in human patients.
To conclude this entry: I predict that the pioneers of CRISPR, Jennifer Doudna and Emmanuelle Charpentier – possibly along with one of the main developers of the technology, Feng Zhang – will win a Nobel Prize. Maybe before 2025?
Here is a short video on how CRISPR works (@BioCenturyTV), together with comments from Jennifer Doudna. And if you want to keep up with all the latest CRISPR papers then the Twitterbot @CRISPR_papers may be of use.
P.S.: Emmanuelle Charpentier is coming to speak at next week’s Crossing Frontiers in Life Sciences conference in Vienna (https://frontiers.univie.ac.at/home/) – I am very excited!