Is the CRISPR Craze a Rerun?

Some years ago there was a basic science discovery that took the biomedical field by storm. Scientists working in a model organism had found a way to selectively target nucleic acids in the cell, shutting down gene expression. There was a ton of hype over the next several years, with everyone imagining the therapies that would start to help patients in no time.

You might think that I am talking about CRISPR; everyone else is, after all. But I am talking about RNAi, which was once touted as the discovery that would revolutionize medicine forever. I was talking to a colleague who is a bigwig in the CRISPR field who was speculating about the future of his field when he said something that shocked me at first. He suggested that CRISPR will not be the revolutionary clinical discovery that some people think it will turn out to be. When I pressed him, he compared it the hype behind RNAi a decade ago. Given this perspective, a couple of questions started to float around in my head. How similar was the hype behind RNAi to that of CRISPR/Cas9 today? Could CRISPR lead to the same letdown?

I did not know much about the RNAi craze–I know RNAi as a handy lab technique, but I never thought of it as a viable clinical treatment–so I went back and did some Googles. RNAi, which stands for “RNA interference,” is a set of cellular systems that cut up RNA and use the pieces to target and attack matching RNA transcripts in the cell. This turns down the expression of certain genes, which can be an effective way of doing genetic experiments in the lab.

It did not take much imagination to dream of how RNAi could be useful in treating human disease. Since plenty of diseases are due to the expression of disease-causing genes, doctors could treat the disease by giving the patient a drug to mobilize the RNAi system against the disease gene.

But in practice, RNAi ended up being difficult to use in patients. Hopes for RNAi therapy peaked during the mid 2000s, and started to ebb during the next few years after human trials showed no real benefit to patients or led to unintended immune responses.

Some people were afraid that RNAi would never live up to its promise. Biotechnology companies shuttered their RNAi research divisions. Human trials slowed down. Luckily, things did bounce back. There are still companies today working on RNAi therapies. It would seem that RNAi was over-hyped, it nearly crashed, then it became what it was always going to be: a therapy with some promise, but no miracle.

Today, CRISPR is just as hyped as RNAi was back then…if not more. CRISPR genome editing is popular science. In many ways, the lay public believes that this will be the century of biology: we will crack the mysteries of aging, we will edit human embryos to eliminate genetic disorders, we will cure all of the diseases. CRISPR genome editing is at the center of these hopes. But there are lessons to learn from the original breakthrough to end all breakthroughs. RNAi was not a complete failure, but we were certainly naive about its potential.

Part of what we got wrong was how unrealistic we were about the limitations of RNAi technology. Living cells have strong negative reactions to double-stranded RNA, which is a necessary step in the RNAi pathway. Delivery systems would be hard to engineer, just like the problems that still plague gene therapy. Finally, there is something that RNAi and CRISPR have in common: off-target effects.

Both RNAi and CRISPR depend on nucleic acids lining up and binding to each other in a pairwise manner before they can have their effect. Since the RNA sequences that bind to targets in RNAi and CRISPR are short and there is quite a bit of nucleic acid sequence in the cell, there is a possibility that you will get your molecule pairing up with an unintended target. It is like taking a short sentence fragment at random from a book and then searching the book for that fragment. You can find the target that you are looking for, but you might also find other perfect or near perfect matches elsewhere in the book, especially when you are searching through a large, complex book.

When these off-target effects happen with RNAi, you could shut down the expression of another gene. If that other gene is important, you might risk harming the cell. The same thing can happen with CRISPR. In fact, CRISPR has the potential to have more dire off-target effects:  CRISPR involves changing the DNA archive, rather than the RNA copy, which can lead to irrevocable changes to the cell.

Luckily, it does seem like CRISPR researchers have taken this to heart. Research into CRISPR’s off-target potential is an active field. I even blogged about a system that might be able to fine-tune the activity of CRISPR/Cas9 with the goal of reducing off-target effects in CRISPR therapy.

To be fair, CRISPR is at least a decade away from the clinic. But there are reasons to be concerned. Scientists have edited human embryos, and ethicists are scrambling to come up with rules to inform how we use this technology. If we learned anything from the RNAi experience, we should carry it over to CRISPR/Cas9. These systems seem to break out onto the scene with a ton of potential and bold claims. Eventually, we might be disappointed. There might be CRISPR trials somewhere down the road that will have to stop, with patients who thought they might be helped instead left wondering what all the hype was about. But if we have learned anything, it is that these systems will change our world. We will end up better off because of CRISPR. We just have to be willing to take the time to figure it out first.

Anti-CRISPRs Could Fine-Tune Genome Editing

Everything needs an off switch. I would have been bankrupt a long, long time ago if I could not turn off the lights in my apartment and C-3PO would have quickly worn out his welcome if he could not shut himself down like he did in Ben Kenobi’s hut. The important thing to remember here is that these things are useful most of the time: light helps me to see but it would not do me any good in the daytime, and C-3PO is like a sassy Google Translate…sometimes too sassy though. And it turns out that even the genome editor CRISPR-Cas9 has an off switch.

Maybe this is the first biology piece you have read in the last three years. If so, you may not know about CRISPR-Cas9 and the genome editing revolution. Commonly referred to as simply “CRISPR” in the popular press, CRISPR-Cas9 is a laboratory method for editing the DNA sequence in a living organism. Throughout the last several years, CRISPR-Cas9 has shown itself time and time again to be a simple and effective way of changing the genome of many different organisms. One group even pursued a controversial study that edited non-viable human embryos, showing that the method can likely be used to edit viable human embryos–as well as setting off a firestorm in the popular press and a lot of ethical hand-wringing within the biomedical community.

The CRISPR-Cas9 system was originally discovered in bacteria, and it functions a kind of anti-viral immune system in bacteria. As I have written before, viruses do their job by injecting a genetic material–DNA in some cases–into a host cell. Some viruses specifically target bacteria. Much like our bodies have evolved defenses against pathogens, bacteria have evolved defenses against viral invaders. This is where CRISPR-Cas9 comes in. Scientists–at a yogurt company of all places–discovered pieces of viral DNA in the genome of a bacterial species that is normally used in yogurt-making. Interestingly, bacteria with these viral signatures were also immune to the corresponding virus. Later work showed that these stretches of viral DNA were actually added to the bacteria’s genome after a viral infection. After that initial infection, the new viral DNA pieces in the bacterium could be made into RNA and loaded onto the protein Cas9. The RNA-Cas9 complex is then free to go bind to DNA that is specified by the RNA, which would be viral DNA in this example. After seeking out complementary DNA from an invading virus, Cas9 performs its molecular function: cutting that DNA into pieces that cannot take over the host cell.

Research on CRISPR-Cas9 has been moving forward at a rapid pace, so I could write exclusively about it and never run out of things to talk about. But a recent published result showed that some bacterial viruses have evolved special proteins to inactivate Cas9, effectively shutting down the CRISPR-Cas9 immune system. It has been known since the middle of the 20th century that protein activity can be controlled by the binding of another molecule. The phenomenon is broadly known as protein regulation, and it is useful because a cell often needs to fine-tune the activity of certain proteins in order to survive. For example, Escherichia coli bacteria prefer to use glucose sugar for energy, but they also can also produce an enzyme to utilize another sugar, lactose, for energy. Interestingly, a lactose molecule can bind to the protein that prevents the production of the lactose-digesting enzyme and allow for the utilization of lactose. Similarly to how lactose can control the protein that shuts down lactose metabolism, scientists recently discovered that a group of viral proteins can shut down Cas9. Importantly, they showed that the “anti-CRISPRs,” as they dubbed the molecules, can bind to the RNA-Cas9 complex and strongly inhibit the DNA-cutting activity of Cas9 in a test tube.

However, the real appeal of CRISPR-Cas9 is not that we can mix it with DNA in a test tube and see DNA cleavage. Instead, we can do all of this in a living cell and cause DNA mutations that can be useful for research or maybe even therapy. If we are going to continue using CRISPR-Cas9 in living cells–perhaps someday therapeutically–we are going to want to fine-tune its activity. Luckily, these same researchers showed that anti-CRISPRs can block CRISPR-Cas9 genome editing in human cells. This result could someday help to avoid “off-target effects” that CRISPR-Cas9 sometimes causes, which are basically just unintended editing effects that could cause more harm than good.