Saturday, January 7, 2017

Original Recipe, Extra CRISPR-y

Today we're not exactly talking about breaking news; CRISPR gene editing has been talked about extensively for a couple years now.  It was named the American Academy for the Advancement of Science's (AAAS) breakthrough of the year in 2015, and articles are coming out constantly with new applications and successes.  I, like most science journalists, find it super cool but pretty complicated, so I wanted to talk about what exactly it is, what has developed recently, and what it might be able to do.

One of the major confusions with CRISPR is that the name is used to describe two different things.  You'll often hear it used to refer to a gene editing technique, but the name for that technique was taken from a phenomenon in bacterial DNA that it exploits.  CRISPR the phenomenon stands for Clustered Regularly Interspaced Short Palindromic Repeats.  It is a complicated name, likely mostly based off of the discoverer's desire to make a pronounceable word, but is actually pretty descriptive of what they are.  CRISPRs are sections of bacterial DNA that contain repetitions of the same sequence of A-T and C-G base pairs over and over again.  These repetitions are separated by segments of foreign "spacer" DNA from viruses and other infectious agents that have attacked the bacteria at some point.  There's a three step process involving CRISPRs: first, the bacterial genome and the viral genome are both cut, and a section of the viral genome becomes a new spacer portion of a CRISPR.  Second, the bacterial DNA with the new spacer undergoes transcription- the process where DNA is made into an RNA template, used for producing more copies of the DNA.  Most importantly, the RNA allows for the third step in the process: it recognizes matching DNA that's introduced to the bacteria, an invader that the bacteria has seen before, and guides enzymes known as CRISPR associated systems, or cas proteins, to cut up and destroy the invader.1  In this way, the CRISPR/cas system is basically a bacterial immune system making antibodies new antibodies when introduced to a new foreign invader, and deploying antibodies when it recognizes a foreign invader.

That's the bacterial phenomenon part.  Scientists have discovered that this system can be altered and utilized to edit genes.  If, instead of invading viral DNA, they use specially designed DNA sequences, they can essentially tell the CRISPR/cas system to cut DNA at any point that they want, allowing them to manipulate an organism's genome by turning on or off very precise genes, removing mutations that may be due to repeats, or potentially allowing other sequences to be inserted.2

This is super cool, but gene editing isn't new- remember having to make glowing bacteria in a high school biology lab?  Even before CRISPR, gene editing has gotten a lot more advanced than just transfecting jellyfish DNA into bacteria, but people are still talking about CRISPR as if it is going to change everything.  It has several advantages over other gene editing technologies.  For one thing, it's cheap.  Compared with having to pay somewhere between $3,000 and $5,000 dollars for specialized DNA segments with enzymes that are kind of a craps shoot, labs can purchase CRISPR kits for around $75 dollars, and they are far more likely to work.  Because CRISPR RNA can recognize sequences of around 20 base pairs, you get more specificity than with some traditional methods that only recognize four base pairs.  Think of it as doing a find and replace in a document.  If you just look to replace "ed" with "ing", you're going to end up accidentally changing the word "seeds" to "seings", when that's really not what you what.  If you look to replace "crawled" with "crawling", you're going to get much more specific results.  CRISPR allows for the same sort of specificity, just with genes.  There are other methods of gene editing that work with even longer sequences of base pairs, and therefore allow for even more precision, but those have the problem listed above- that a special protein has to be created for everything you want to use it on, and there's a lot more room for error.  This is because the CRISPR/cas system comes with its own enzyme for cutting DNA.  Instead of having to have a cleaving enzyme, guiding RNA, and a system for healing the spliced DNA, all of which have to be correct, CRISPR eliminates those difficulties by essentially being a self contained package, making it cheaper, faster, and more efficient.  It also has the advantage of allowing you to target more than one sequence of DNA, allowing you to manipulate multiple genes at one time.3

With most new scientific techniques, people might talk a big game about the implications, but the actual applications are probably a least a dozen years off.  Not quite so with CRISPR.  It first started being looked at for gene editing in 2012, and in the four years since, over 1,000 papers have been published.  Research is moving so quickly that it is already coming up with solutions for some of the early limitations.  For example, scientists have already found an alternative enzyme with CRISPR sequences that cuts DNA without needing a specific DNA sequence nearby.  By tethering cas to an enzyme that doesn't just cut DNA but actually converts one base to another, an A to a C for example, we're moving forward on not just cutting DNA and removing segments, but actually changing DNA.  Originally, the CRISPR/cas complexes were too large to put into a lot of the vehicles that are commonly used to introduce them to a genome, but a possible solution for that has recently come along as well.4

The really amazing question with CRISPR is what being able to precisely edit genes means practically.  Here we get into the really cool stuff.

Disease research: The most common way of studying human diseases in animals is to use knockout mouse models.  These are lines of mice that have been bred to not have a certain gene that we think plays a role in a disease- obesity, cardiovascular disease, depression, etc.  We can use knockout mouse models to study behaviors caused by a gene, potential therapies, and the interactions of genes with environment, among other things.  CRISPR allows the possibility of creating new knockout mice by allowing us to a) target new genes, b) develop a knockout mouse line in a period of weeks instead of months, c) saving money, time, and several generations of mice's lives.5  These knockouts could also potentially have alterations in several genes, allowing for more complex and accurate models.6  CRISPR is being looked at to create primate models of disease that even more closely mimic human diseases, a path that hasn't really previously been open given the ethical concerns of creating a line of genetically engineered primates.7

Disease treatment: CRISPR has already made its way into clinical trials for disease treatment.  China has used it in non-viable human embryos to alter the genes that code for β-thalassaemia, a potentially fatal blood disorder.  Only 33% of the embryos survived and were successfully spliced, at which point they stopped the experiment until CRISPR research matures.8   A second group in China has also used CRISPR in non-viable embryos, this time attempting to splice in a gene that codes for HIV immunity.  This experiment was only successful in 15% of embryos, but these two experiments serve as a proof of concept for the potential application of treating genetic disorders or granting immunity from disease before embryo implantation.9  Obviously there are huge ethical controversies involving this research, which is another post for another time.  On the more immediate front, in October of this year the first trial of CRISPR in a living, adult human began.  Doctors in China delivered CRISPR modified cells into a patient with aggressive lung cancer; they have not released any followup reports on the success of the trial.  A similar trial is approved to begin in the US in 2017 to treat various cancers.  The idea of these studies is to use CRISPR to target mutation in genes that are causing cancer, attempting to stop the spread and development.  From what I can tell, this isn't a treatment for any current tumors, more that it will keep cancer from growing, spreading, and returning.10  Another very interesting disease treatment application is using CRISPR to treat naturally occurring viruses in pigs.  Although pigs are some of the most similar animals to humans biologically, these viruses have kept them from being widely used as hosts of transplant organs.  By addressing the virus issue, porcine organs have a much lower chance of rejection and may become more viable for human transplant.11  In mouse models, scientists have also used CRISPR to treat retinitisa pigmentosa, a cause of blindness,12 to correct tyrosinemia, a genetic liver disorder,13 and to improve cholesterol.14

Agriculture:  There are a couple big areas utilizing CRISPR in agriculture.  The first is the obvious: genetically modified plants that increase yield, nutritional content, and photosynthetic efficiency, faster and with more precision and efficiency than traditional GMO crops.15   Basically, more crops that provide more nutrients for us.  All good things, but you still have a lot of the GMO arguments currently going on (another post for another time).  Plants can also be modified with CRISPR to give us more options for plant based drugs and vaccines.  We can use plants to do the work of creating proteins and metabolites for us, and CRISPR can both increase our knowledge of these systems and, again, increase specificity.16 

Petrochemicals: I love this one because it goes beyond the obvious applications of "genetic engineering".  Fossil fuels are a problem.  Burning cleanly, having enough, the damage on the environment.  We really need to come up with efficient alternate energy sources, and CRISPR offers us a chance at just that.  By engineering bacteria, yeast, and fungi to control the hydrocarbons they produce, there's the opportunity for a lot of innovation in producing biofuels, plastic polymers, and adhesives.17

These applications don't even begin to get into controlling reproductive and feeding drives of animals, genetically modifying mosquitoes so they don't carry malaria, selective breeding of livestock, creating antibacterials and antibiotics that are more targeted and don't carry the risks of making immune bacteria, and a host of other applications.  What is truly amazing about CRISPR, besides the wide range of impacts, is the speed at which these developments are happening.  Basically all of the aforementioned breakthroughs have happened in the last three to four years,outpacing just about every other technology by miles.  From the first paper coming out in 201218 saying the CRISPR/cas system could potentially be manipulated to clinical trials in humans in a four year span is insane when it comes to science.  Keep an eye on CRISPR in the next couple years.  This one really does have a the promise to change a lot of things, and change them sooner rather than eventually.

1 comment:

  1. Thanks for the detailed information. My grandpa is having multiple sclerosis. It's a very complicated disease but as doctors are assuming she's getting improved everyday with the help of multiple sclerosis treatment.