Cas9 cystal stucture

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.

 

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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.
https://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.
https://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.
https://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

 

 

 

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