Gene Drives: so you want to change the world!

By Rich Feldenberg:
Want to change the genetic landscape of whole populations and ecosystems? Tired of having to do it the old fashioned way by genetically engineering one organism at a time? Well now there’s Gene Drive! The fast and efficient way to spread your desired genetic design! Just send $19.99 plus shipping and handling for your Gene Driver Kit today!

If the “Fake Advertisement” in the paragraph above, made it sound as though the Gene Drive concept is some crazy kind of internet scam that is to good to be true, actually nothing could be further from the truth. Well no, you can’t just send in money for a gene drive kit yet, but it turns out that gene drives are real, they’re awesome, they’re controversial, and they can in principle, change the gene pool of an entire population of an organism. In fact, this method of gene editing is so new that very few experiments have even been done, and its founder, Kevin Esvelt, feels that the technology is so powerful that he wants to put a halt on experimentation until society can come together and discuss whether we collectively feel this is an area of science we should pursue, not just one that we can pursue. To understand gene drives we first have to remind ourselves of how the CRISPR-Cas9 system works, which I reviewed in an earlier Darwin’s Kidney post.

Briefly, the CRISPR-Cas9 system is a new and powerful gene editing technique that can be used in living organisms. This system is found naturally in many bacteria, as part of their immune defense mechanism against viral attack. There are two major parts to the system. The first is a guide RNA and the second is the Cas9 enzyme. The guide RNA is a small strand of RNA (somewhere around 20-40 base pairs in length). When the guide RNA finds a perfect base pair match with a DNA strand somewhere in the cell, the Cas9 enzyme cuts that piece of DNA. In the case of bacteria, this allows the them to match one of their guide RNAs to a sequence of DNA from an invading virus, then cut the viral DNA, which disables the virus from taking over the bacterial cell. The guide RNA came from a previous viral attack that the bacteria survived, and when the bacterial enzymes chopped up the invaders DNA into small bits, some was incorporated into CRISPR so that exposure to that same virus the next time would quickly result in recognition by the bacteria – an immune system! In the last few years scientists have discovered how to make guide RNA for any desired gene, and along with the Cas9 enzyme, can then “snip out” the gene or any piece of DNA in question. This can be used to silence genes, or can also be used to replace genes if the cell has access to a DNA sequence that can fill the gap left by the Cas9 enzyme. This may turn out to be a great way to cure genetic diseases through gene therapy.

Gene drive systems, take this concept a step further. Gene drives rely on the gene editing to take place in germ line cells versus somatic cells. Germ-line cells are the cells that will become egg or sperm, and will be used to create new organisms through sexual reproduction. Somatic cells are all the other body cells, such as skin, kidney, brain, pancreas, etc. If a gene is edited in a somatic cell, that change will effect the organism in whom the change was made, but would not be passed down to the next generation.

As an example, lets say you want to be able to provide gene therapy for a genetic disease such as Nephrogenic Diabetes Insipidus (NDI). This disorder is X-linked, meaning that the gene is on the X chromosme. Since males have an X and Y chromosome, with the X coming from their mother and the Y coming from their father, if the mother’s X chromosome has the mutant gene for NDI they will have inherited the disease, which leads to the kidneys inability to regulate water loss. People with this disorder can die of dehydration because even when dehydrated they continue to produce too much urine. A female, having two X chromosomes, one from her mother and the other from her father, might be a carrier for NDI if her mother’s X had the mutant NDI gene, but she still wouldn’t develop the actual disease since her normal NDI gene from her father’s X chromosome will compensate.

In principle you could use the CRISPR system to edit the defect gene, so that the male patient with NDI can now regulate water loss through the kidneys normally. There is still no way to really do this yet. You would need to deliver the CRISPR-Cas9 system, to the appropriate kidney cells of the affected individual. At the present time, a way to target and deliver the system is still not available, but if it could be delivered to the kidney cells it would excise the defective DNA. The cells own repair mechanisms will then look for a replacement to fix the DNA break made by the CRISPR-Cas9. If the normal gene was also delivered to the cell it will be incorporated into the place where CRISPR-Cas9 made the break. This will result in having removed the defective disease causing gene and replaced it with the normal healthy gene, and should therefore cure the disease – Nephrogenic Diabetes Insipidus kidney disease in our example. However, even if this could really work – its never been tried yet for this disease – but was unsuccessfully attempted for Hemophilia, the cured individual would still be able to pass the disease on to their children. The reason is that only the kidney cells were altered, and not the germ-line cells.

Gene drives, on the other hand, effects the germ-line, but they have an even bigger, more ingenious twist to their potential to alter future generations. With gene drives, in addition to supplying the new gene, the genetic code for more CRISPR-Cas9 is also inserted into the target genome. So here is how it might work. Let simplify the example by calling the two alleles of the gene (one allele comes from mom and the other from dad) as Normal and Engineered. It could be any gene in the genome that you’re interested in, such as the gene for making insulin or for making neurotransmitters in the brain, or transcription factors that tell more genes what to do. In this example we want the Engineered gene to take over because it has some trait we have engineered for it that we find desirable. It could be to fix a defective gene or it could be to give the organism some new property. We’ll get to some examples of new properties shortly.

 

The first step might need to take place in the lab when the organism at its earliest stage, the fertilized egg. You place the CRISPR-Cas9 into the fertilized egg with guide RNA that recognizes the Normal gene. You also place the Engineered gene in the cell to replace the Normal gene once Cas9 has cut it. The cell will now have the Engineered gene as part of its entire genome. This will effect both somatic cells and germ-line cells since the fertilized egg will continue its job of dividing into more and more cells, which will eventually become all the cells of the body. Eventually this organism will develop into an adult and find a mate to produce more offspring. The offspring will have an approximately 50% chance that the Engineered gene will be passed on to the the next generation. That is because each offspring will get one copy of Engineered gene from our genetically modified organism and the other gene from its mate, which would carry the Normal gene version since it was never modified. So this is where the special ingenious twist comes in!

Not only does the gene you inserted into the fertilized egg contain the DNA of your engineered gene, but it contains the DNA for making a CRISPR-Cas9 system, as well. This CRISPR-Cas9 is hidden somewhere in the middle of your Engineered gene so that the cells DNA repair enzymes don’t recognize it as being novel to the cell. They only recognize the ends that need to fit in the space that Cas9 cut out. So in this way, the gene we pasted into the genome is Engineered-CRISPR-Cas9. Now when the cell transcribes that gene the CRISPR-Cas9 is also transcribed which leads to a guide RNA and a working Cas9 enzyme. The guide RNA will then match to the Normal gene and Cas9 will cut it. This is important because when the genetically modified organism mates with a wild type organism the offspring will have one Normal gene from the wild type and one Engineered-CRISPR-Cas9 gene from the genetically modified organism. CRISPR-Cas9 then gets transcribed, seeks out the Normal gene, and replaces it with the Engineered-CRISPR-Cas9 gene, so the offspring actually ends up with two copies of the Engineered-CRISPR-Cas9 gene. In this way the rate of transfer of the Engineered gene, to successive generations, goes from 50% to 100%. The Engineered-CRISPR-Cas9 gene effectively edits every other allele that matches its guide RNA, turning it into the Engineered gene. Now the gene can spread rapidly through a population because the odds are always in favor of this gene being passed down to all offspring. See the excellent Mosquito chart in the following article I’ve linked to in order to get a visual on how the inheritance would be effected.

It should be pointed out that this is only effective for organism that reproduce sexually. Asexually reproducing organisms (such as bacteria) won’t be influenced by this mechanism. For organism that have short generation time this is ideal. One proposed problem that gene drives might be able to solve would be in the fight against malaria. Malaria kills millions of people each year (see Darwin’s Kidneys article: Diseases with an Upside). If mosquitos were released into the wild, that were engineered to have a malaria resistant gene and also the CRISPR-Cas9 system, then that gene would spread rapidly throughout the mosquito population. The result would be malaria resistant mosquitos and possibly an end to suffering and death in many parts of the world due to this parasitic infection.

There’s no guarantee, however, that the malaria organism – Plasmodium – would not find a way to evolve around the mosquito’s malaria resistance given enough time. There is also no guarantee that the malaria resistant gene might not somehow decrease the “genetic fitness” of the mosquito making them less likely to survive and reproduce. Mosquitos would be an ideal organism for this type of engineering, however, since they have a rapid generation time, so within several years to decades a gene system of this type could theoretically pass to all members of the population. Humans, on the other hand, reproduce slowly so a gene drive in humans would probably take hundreds of years to spread through the population. Still, you could imagine an attempt to eliminate many genetic diseases completely from existence by using gene drives that over the course of centuries might be effective. One could also imagine the ability to produce a civilization of future generations of humans that are more intelligent, more rational, less violent, more empathetic, and so on, if the genes involved in producing those traits could be identified. It is harder to imagine, however, that society as a whole would ever agree to such a mass alteration of the human genome – creating something beyond human – by directing human evolution in a desired direction. Its too early to know if such changes to the human genome could even be done safely without creating damaging consequence that are impossible to predict. I’m not necessarily advocating for changing the human race for the better, but more just advocating for discussion of the potential positive and negative effects might result from such grandiose dreams.

Because the implications for gene drives are so powerful and large scale, there is currently a call for a hold on research until the ethical considerations can be more fully considered. I think this seems wise at our current state of understanding. Changing an ecosystem could have unforeseen consequences. There may be ways to alter some behavior in organisms with gene drives that would not necessarily eliminate those organisms from the ecosystem – and so may have a mild impact on the ecosystem as a whole. For example, one could engineer a pest to dislike the taste of a crop that it normally damages, and therefore protect the crop without the need for as much pesticide use. The pest is now no longer a pest, but remains in the ecosystem where it can feed on other plants and remain part of the normal food chain for other organisms. Could gene drives be used to engineer plants to more efficiently remove CO2 from the atmosphere, and combat global warming while increasing crop yields?

Gene drives are an exciting new method of changing the genetic makeup of populations of organisms. Whether they will be used to prevent diseases like malaria from killing so many or making crops less prone towards pests and therefore reducing the amount of insecticides released into the environment, is up to society at large to decide if we are ready to pursue such far reaching technology. My hope is that we may find ways to safely use gene drives to improve life on planet earth for ourselves and our fellow species.

References:
1. “Genetically Engineering Almost Anything” by Tim De Chant and Eleanor Nelson, Nova Next. July 17, 2014.
http://www.pbs.org/wgbh/nova/next/evolution/crispr-gene-drives/
2. “Gene Drives and CRISPR could revolutionize ecosystem management”, by Kevin Esvelt, George Church, and Jeantine Lunshof; Scientific American Blog. July 17, 2014.
http://blogs.scientificamerican.com/guest-blog/gene-drives-and-crispr-could-revolutionize-ecosystem-management/
3. Gene Drive Wikipedia: https://en.wikipedia.org/wiki/Gene_drive
4. “Gene editing in Humans”; Neurologica blog by Steven Novella; Nov. 19, 2015
http://theness.com/neurologicablog/index.php/gene-editing-humans/
5. “CRISPR: what’s the big deal?”, Darwin’s Kidney blog by Rich Feldenberg. Nov. 28, 2015.
http://darwinskidneys-science.com/2015/11/28/crispr-whats-the-big-deal/
6. “Can we genetically engineer Rubisco to feed the world?”; Darwin’s Kidney blog by Rich Feldenberg.
July 22, 2015.
http://darwinskidneys-science.com/2015/11/28/crispr-whats-the-big-deal/
7. “Diseases with an upside”; Darwin’s Kidney blog by Rich Feldenberg. July 29, 2015.
http://darwinskidneys-science.com/2015/07/29/diseases-with-an-upside/
8. “Live at the NESS: New Dilemmas in Bioethics”; The Rationally Speaking Podcast. April 24, 2011.
With Massimo Pigliucci and Julia Galef as hosts.
http://rationallyspeakingpodcast.org/show/rs33-live-at-necss-new-dilemmas-in-bioethics.html

9. “Sculpting Evolution”; website of Kevin Esvelt, PhD.  Founder of gene drives.   http://www.sculptingevolution.org/kevin-m-esvelt

 

 

 

CRISPR: what’s the big deal?

By Rich Feldenberg:
In the last couple years there have been a growing number of mainstream media stories (like this recent CNN article) highlighting a new molecular biology technique that is revolutionizing the way scientists conduct genetic experiments, and may soon make the holy grail of medicine (gene therapy) possible. It certainly seems far from usual for the media to be overly concerned with a technical method of scientific investigation, but CRISPR has caught the attention of scientist and non-scientist alike due to its huge potential to change the research landscape. This article will discuss what CRISPR is all about, what it does in nature, how scientists are using it in the lab in place of older more traditional techniques, and what its future potential might be to cure diseases that are now incurable.

CRISPR (pronounced like Crisper) is an acronym for Clustered Regularly Interspaced Short Palindromic Repeats. Yeah, CRISPR is much more fun to say. It is a naturally occurring segment of DNA found in many prokaryotes. Prokaryotes are single celled organisms that lack a nucleus and other internal structures that more advanced eukaryotic cells contain. The prokaryotes include bacteria and archaea, and CRISPR has been found to occur in about 40% of bacteria and 90% of archaea sequenced so far.

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The CRISPR DNA is organized in a particular way. It is made of short lengths of repeating DNA basepairs that are separated by regions of seemingly random DNA, known as spacers. Both the repeating regions and the spacer regions are on the order of 24-48 base pairs in length. These repeating structures were first discovered in the DNA of E.coli (a common bacteria found in the intestines of humans) back in 1987, but their function was not known at that time. It was not until 2005 that their function began to become understood, thanks to bioinformatics. Bioinformatics is a computational biological approach to problems in molecular genetics. Using computer programs to search and compare genetic databases, it was found that the spacer portions of CRISPR exactly matched portions of DNA from bacteria infecting viruses and plasmids. That lead to the realization that CRISPR serves as a kind of immune system for prokaryotic cells.

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Basically what happens is that when a virus infects a bacterial cell, the cells first line of defense includes nucleases (enzymes that cut up DNA) that are released in an attempt to destroy the invaders genetic code. The majority of cells infected will not survive, but in the rare chance that it does survives the viral attack, nucleases then cut up the virus DNA into small parts, some of which become the spacer segments in the CRISPER complex of the bacterial DNA. Next time that the same type of virus infects the bacteria, the bacteria can quickly identify it based on the DNA match between its CRISPR spacer segment and the viral DNA. Enzymes called Cas (for CRISPR associated) are nucleases that will associate with the spacer sequence. They cut the viral DNA at the specific place where the match occurs. This increases the chance for the bacteria to survive the viral attack and confers a kind of immunity to the cell.

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It has been shown that bacteria that contain CRISPR are much more resistant to virus that have DNA sequences contained in their CRISPR spacers, and if those spacers are removed from their CRISPR segments, they lose that resistance. There are certain Cas enzymes that can continue to add new spacers each time they are attacked with new kinds of viruses, and if these types of Cas enzymes are defective or absent, the bacteria can still defend against attack with familiar viruses but are unable to acquire immunity to new ones.

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This certainly seems like good new for bacteria, which are continuously under attack by viruses day in and day out. In fact, it has been estimated that viruses kill 40% of the bacteria in the oceans each and every day. It didn’t take long for scientists to realize that CRISPR could provide an amazing and precise genetic tool. Since the spacer sequence can recognize very specific regions of target DNA and Cas9 (the particular Cas enzyme that is attached to the spacer) can then carry out a seek and destroy mission of the target DNA. It is like the delete key in a word processor program.
It was found that the target DNA didn’t have to be just viral DNA for the process to work. If DNA from a bacteria, animal, plant, fungus, or apparently any organism was inserted into the spacer sequence of CRISPR-Cas9 complex, then a genetic modification could be easily made to that organism. Using CRISPR, genes can be easily and cheaply edited. This system has set in motion a new revolution in molecular biology that has not been seen since PCR (polymerase chain reaction), a technique to amplify DNA was first introduced in the 1980s.

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So what can CRISPR really do in the lab? It can be used to delete specific genes or segments of genes. All you need is a copy of the basepairs that correspond to where you want your deletion in the target organism, and with that and your Cas9 it will find the correct place on the DNA and then cut the DNA at the precise location. In that way you can remove a gene. You can also insert a gene as well. This is done in a similar manner. You have your target DNA picked out and inserted into your CRISPR system, but in addition you need the gene, or segment of DNA you want to insert placed into the cell, as well. The cell’s own repair mechanism will detect the damaged DNA and attempt to repair the break with the added gene. In this way it acts like the cut and paste function of your word processor.
By removing the DNA from the target you are making an irreversible change to the genome of that organism, but it is also possible to use CRISPR to make reversible changes too. This is done by using a defective Cas9 enzyme. The spacer DNA sequence will still seek out and find the desired region of genome, but due to defective Cas9 the DNA will not be cut out. The spacer sequence will still attach to the DNA and block transcription, so is effectively turning off that gene.

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These sorts of techniques can be used to make “knock out” organisms – organisms that lack a particular gene. Knock out mice, for example, are an indispensable tool for understanding how certain genes function in a whole creature, and how mutations in those genes lead to certain diseases. Genes can also be turned on by CRISPR by combining the CRISPR complex with a promotor – a regulatory element that tells the cell to begin transcribing a particular gene.

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This is now allowing labs around the world to investigate disease causing mutations, including cancer biology, at a much faster rate. The techniques are considered relatively easy to use and master, and much less expensive than traditional molecular techniques to achieve similar results.
CRISPR has already been discussed as a potential therapeutic medical intervention, although, the ethics of genetic engineering are still being hotly debated. There was a attempt to use CRISPR to cure the disease hemophilia by a group of Chinese researchers, although they were not successful it is likely only a matter of time before progress along these lines would make similar trials more effective. Right now it is considered by most to be too new of a therapy for clinical medicine, and even if it could be done, some consider tampering with the human genetic code too dangerous. This might especially be true for genetic alterations that would be passed on to subsequent generations beyond the individual being treated. Some worry that it will go beyond just curing disease, and be used by the wealthy to create “designer babies”. Perhaps couples that want taller, stronger, or smarter children would be able to engineer their children to be so. Would that create an even wider divide between the haves and the have nots? These are certainly questions that should be addressed and discussed, but I do hope that fear won’t prevent this technology from reaching it full potential to treat genetic disease.

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Would it really be so bad to have future generations of humans that are more disease resistant (perhaps less prone not only to heart disease, diabetes, and Alzheimers, but less prone towards depression, addiction, or apathy)? What about a future of humans that are more intelligent, more rational, less violent, more compassionate and empathic? Only by being properly informed can we make the best decisions as a society about how to best use such technology.

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References and other reading:
1. CRISPR wikipedia article: https://en.m.wikipedia.org/wiki/CRISPR
2. CRISPR interference wikipedia article: https://en.m.wikipedia.org/wiki/CRISPR_interference
3. “CRISPR/Cas9-mediated gene editing in human triproneulcear zygotes”; Protein & Cell; May 2015, Vol6, Issue 5, pp 363-372. http://link.springer.com/article/10.1007/s13238-015-0153-5
4. “Molecular Biology of the Gene”; 7th Edition, James D. Watson, Cold Spring Harbor Laboratory Press; 2014. Pages 706-712.
5. http://www.geneticliteracyproject.org/2015/06/25/ethical-and-regulatory-reflections-on-crispr-gene-editing-revolution/
6. http://www.nature.com/news/ethics-of-embryo-editing-divides-scientists-1.17131
7. http://www.cnn.com/2015/10/30/health/pioneers-crispr-dna-genome-editing/
8. Neurologica blog article on CRISPR: http://theness.com/neurologicablog/index.php/gene-editing-humans/
9. “Is Bad Luck Really a Diagnosis?”, Darwin’s Kidneys blog: http://darwinskidneys-science.com/2015/06/14/112/