CRISPR: The almost unpronounceable discovery of the century that everybody is talking about. If you do a simple Google search you’ll get over 4 million results regarding CRISPR. Moreover, there’s a high chance that this acronym will appear in the Nobel’s prize very soon. But do you know why it is such a relevant discovery? Do you know that there is a huge ongoing fight over CRISPR patents? And better yet, did you know that this patent war might change its tune completely due to an endonuclease named Cpf1?
Like any book lover who read Aldous Huxley’s ‘Brave new world’ or any film lover who saw Ethan Hawke and Uma Thurman in ‘Gattaca’, I was scared about the possibility of genetic manipulation in humans. But then, when you read on how the CRISPR technology could be used to cure cancers, kids with sickle cell anemia or people with the terrible Hungtinton’s disease, as well as hundreds of other genetic diseases by replacing the aberrant genes that cause these diseases for ‘good’ genes, then it is perfectly normal to have second thoughts about it. In any case, for having an opinion on this issue, what is important is to be informed on what this new genetic tool is about and what are the latest discoveries surrounding it… and that is the purpose of this post.
CRISPR, the archaea and bacteria’s defending weapon against virus
At the very beginning of the 1990’s in Spain, Francisco Mojica and his group was studying how could archaea adapt and survive in extremely saline environments. When analyzing a chromosomal region that they thought could be related to this adaptation, they noticed a very peculiar genomic structure that rapidly caught their attention: an information that repeated several times, like a pedestrian zebra crossing. In 2003, they found that these sequences constituted an acquired immune system that allowed these microorganisms to fight against invaders such as viruses. The trick of this immune system, present in archaea as well as in bacteria, is that it allows viral DNA to be integrated into the chromosome and used as a bar code to recognize and fight against virus whose DNA was ‘stolen’ in the first place.
But how does this genomic sequence work as a defending weapon? When archaea and bacteria undergo a viral attack they detect the foreign DNA that gets injected into the cell and integrate short pieces of it into the CRISPR locus within their chromosome. This integration does not occur randomly, but instead specifically as ‘spaces’ between short palindromic repeats (that’s why these foreign bits of DNA are also known as “protospacers”). Interesting thing to keep in mind about palindromic sequences is that when expressed on a single strand of RNA, they get to fold up on itself and can serve as kind of a handle for proteins to grab on to, as in the case of Cas endonucleases (Cas stands for CRISPR-associated proteins).
These foreign bits of DNA are further inherited through cell division and upon a second viral infection they are transcribed into pieces of RNA that are subsequently used together with Cas proteins to form an interference complex. This complex uses the information in these RNA molecules to base-pair with the matching sequences in viral DNA and Cas proteins, which grab on to these RNAs, then breaks down the viral DNA thus preventing infection from going on.
Besides its mechanism, what is amazing of CRISPR is that elements of this system can be transferred to any cell, including human cells. The purpose of this transference is that this system can be programmed to cut wherever we want it to cut, that means we can edit the genome information. Once the cut is done, the DNA-repair system of the cell tries to repair it and by doing it, it introduces modifications. Moreover, these modifications or changes that can occur randomly, can be directed by introducing a template. In other words, we will be saying to the cell: “look, there’s going to be a cut in your DNA right here and you will be using this information that I’m giving to you to introduce this new information in that place”.
By changing the genetic information of any living cell, we can study its function, reproduce genetic defects in mice and try to find drugs against them. Also, in a more futuristic application, we could modify genetic aberrations directly in humans, for example to eliminate viral information that keeps stuck within our cells upon a viral infection, like with AIDS or HERPES virus, thus eradicating the infection and even preventing a cell to be infected.
Engineering the CRISPR system to be programmable DNA scissors
Jennifer Doudna, a researcher from UC Berkley, was interested in understanding how RNA molecules are used to regulate expression of proteins from the genome. And so, the CRISPR system seemed like a very good example of this. In 2011, she started collaborating with microbiologist Emmanuelle Charpentier, from the Max Planck Institute for Infection Biology, who was interested in bacteria that are human pathogens, particularly in Streptococcus pyogenes. What was interesting in this bacteria was, that it has a CRISPR system and that there was a single gene encoding a protein known as Cas9, which had been known genetically to be required in the CRISPR system, although nobody knew what the function of that protein was at that time. So together, they started to test it.
They found out that Cas9 has the ability to interact with DNA, generate a double-stranded break in the DNA at sequences that match the sequence in a guide RNA (crRNA). Moreover, this guide RNA base-pairs with a second RNA named “tracer RNA” (tracrRNA) forming a structure that recruits the Cas9 protein. So those two single RNAs and a single protein are what in nature is required to recognize and destroy viral DNA to prevent infection.
But Doudna and Charpentier’s major contribution is that they generated a simpler CRISPR system by linking together the two RNA molecules and getting a simplified system consisting of just one guiding RNA and one DNA-cutting protein. In other words, they got a DNA cutting enzyme that could be programmed with a short piece of RNA to cleave essentially any double stranded DNA sequence.
In 2013, Feng Zhang from the Broad Institute of MIT and Harvard, successfully adapted the CRISPR-Cas9 system for genome editing in eukaryotic cells (Cong et al., 2013). Zhang and his team engineered two different Cas9 orthologs (from S. thermophilusand S. pyogenes) and demonstrated targeted genome cleavage in human and mouse cells. They also showed that the system could be programmed to target multiple genomic loci, and could drive homology-directed repair.
What the whole CRISPR patent war is about
The origins of the patent dispute began with an issue of money. Jennifer Doudna’s original patent application was filed in March 2013, while Feng Zhang filed his own in October 2013. Despite of filing later, it was Zhang and the Broad Institute who were granted more than a dozen CRISPR patents for genome editing. WHY? Because Zhang paid extra for an accelerated examination of their key patent application, resulting in him getting his first patent in April 2014.
The University of California (UC) challenged the United States Patent and Trade Office (USPTO) decision, which currently has to decide the billion-dollar patent battle that holds the potential of editing human genomes and curing genetic diseases. UC’s attorneys alleged that all of the Broad’s CRISPR patents were obtained through “inequitable conduct”, stating that Zhang “never had or made use of “tracrRNA”, that is an essential component of the CRISPR-Cas9 genome-editing system “in any of the submitted experimental data and results”. “It is believed that Broad withheld or misrepresented material information with the intent to deceive the USPTO” into believing that Zhang had accomplished more than he actually did, UC wrote in its motion. On the other hand, the Broad’s attorneys deny those “unfounded” allegations and also make one crucial argument: that UC’s claims are unpatentable because they overreach what Doudna actually accomplished. As George Church from Harvard said, the spark that Doudna and Charpentier had, was that CRISPR would be a programmable cutting device, but getting it to do precise editing, via homologous recombination, was a completely different thing.
As for the patents, Doudna’s application claims described only “genetically modified cells that produce Cas9” and “Cas9 transgenic non-human multicellular organisms”; Zhang’s application, unlike Doudna’s, specifically contemplated adapting CRISPR in eukaryotic cells.
Another important issue in this whole patent drama is that when Doudna filed her patent application, the law at the time stated that patents go to the first to invent something. But after her application filing, a new law came into effect, which states that patents go to the first to file. Although Zhang filed months after the first-to-file rules came into effect, he claimed a December 12, 2012 priority date under the old first-to-invent rules. So for now, both sides will have to focus on presenting evidence from publications and notebooks trying to show they first uncovered their discoveries on CRISPR. Doudna and Zhang should soon meet at Court and might be required to take the stand. We’ll see how things turn out. What’s clear, is that CRISPR discoveries have attracted a lot of money, public recognition and pride.
The discovery of Cpf1, the protein that can change this whole scenario
In September 2015, Feng Zhang published a finding that could change the whole CRISPR patent war. It showed that a new protein other than Cas9 had been discovered to perform the gene editing, a protein called Cpf1. Because Cpf1 correspond to a new DNA-cutting protein, it is out of the scope of the CRISPR-Cas9 patents.
Zhang and his team discovered Cpf1 by screening thousands of CRISPR systems in different bacteria in order to find enzymes that could be used in human cells. Cpf1 was finally found in the genus Acidaminococcus and Lachnospiriceae. The newly discovered CRISPR-Cpf1 system is a new class CRISPR system, able to cut the target DNA substrate in human cells under the crRNA’s guidance. Moreover, Cpf1 itself is a sequence-specific RNase, which is the only discovered nuclease that has both nuclease sequence specificity and the viability of DNase and RNase. Some of the differences between Cpf1 and Cas9 are shown below.
This discovery is very important for the commercialization of the CRISPR technology which is starting to take-off, specially for the following companies: the CRISPR leading company Editas Medicine (connected to Zhang), Doudna’s biotech Intellia and Charpentier’s Crispr Therapeutics.
The latest discoveries of Cpf1
In 2016, two very important publications of Cpf1 came out in Nature. The first one revealed details about its structure and mechanism, and also provided the structural basis for its improvement, which could help the Cpf1 system to become the specific and efficient new gene editing system. This work used biophysical and structural biology to reveal in detail how Cpf1 recognizes and processes the precursor crRNAs, and also underscores the catalytic and structural flexibility of Cpf1.
Cpf1 and crRNA can be seen as a real couple, before and after marriage. Cpf1 is in a loose and flexible conformation, but after its combination with crRNA, Cpf1 has an obvious conformational change, into a compact triangular structure.
The ‘love element’ between these two, as seen by structural data, is an (Mg(HO))2+ ion, with such a critical function that stabilize the conformation of crRNA and stimulate the activation of Cpf1. Moreover, it could be directly involved in the catalytic reaction of the substrate. These observations were corroborated by a powerful biophysical method named Microscale Thermophoresis (MST) that can measure molecule interactions. MST showed that the binding affinity between crRNA and Cpf1 was reduced nearly 50 fold in the presence of the chelating agent EDTA.
The second publication focuses on the crRNA-processing activity of Cpf1 and demonstrates that the enzyme, besides cleaving the foreign DNA, is responsible for the cleavage of pre-crRNA, thus yielding the mature form used for homology-directed cleavage of DNA targets. Furthermore, this study identified three amino acids essential for crRNA processing.
Science has, without a doubt, entered an era where money and recognition have gained power over scientific discoveries. I can’t imagine Leonardo da Vinci or Isaac Newton getting involved in this kind of heated discussion on who gets credit for science achievements, but then again this is only a guess. What’s important besides all the conflict though, is that good science is being made, that amazing results are coming out, that the future is here and we need to be informed on such important breakthrough discoveries and its applications, as in this case, on human genetic manipulation.