Genetic Engineering

CRISPR-cas9, The Promise and the Peril

 

Nearly everyday some new advance in science and technology is heralded in newspapers and science journals; from the detection of gravitational waves and the discovery of the Higg’s boson/field in physics to the discovery of the suite of BRACA genes that cause breast cancer to the now worldwide controversy caused by the development of the CRISPR-cas9 gene editing technique in biology/medicine. It is the latter, the CRISPR-cas9 gene editing technique, which holds perhaps the greatest promise for changing and improving the human condition and the greatest risk for destroying both the fabric of society and the biological basis for life. The stakes are huge and the dangers from unintended consequences and perhaps even the intended consequences can’t be overstated.

What is CRISPR-cas9?

The simple answer is that it is a gene editing tool that is very, very specific(though not without errors that will be discussed in detail later). The tool allows researchers to potentially target specific genes, such as the sickle-cell gene that causes sickle-cell anemia, excise or cut out that gene, and eliminate it completely or replace it with a healthy gene. The details of how this is done is where the devil is lurking that has caused all of the controversy and the convening last year of The International Summit on Gene Editing attended by the world’s leading researchers and ethicists to discuss the ethics of using the CRISPR-cas9 gene editing tool  and to develop guidelines for future research.

To fully understand the answer to the question of what is CRISPR-cas9, some basic molecular biology is needed. Everyone by now has heard of DNA. TV cop shows and sensational murder cases have all popularized the term. DNA(deoxy-ribonucleic acid) along with RNA(ribonucleic acid)is present in every living cell(and viruses) and serves as the code for millions of variations or forms of life. How does it do this? DNA is composed of four base pairs, which you have probably seen represented as A,T,C,G. These letters stand for the chemical components(bases): A for adenine, T for thymine, C for cytosine and G for guanine. Within a molecule of DNA T pairs with A(chemically bonds with) and C pairs with G. In RNA, thymine is replaced by another base, uracil.

A DNA molecule forms a double helix as a result of the chemical bonds formed between the base pairs on two separate strands. Here are two images of what it looks like. One image is a highly abstract model(A), the other shows the actual chemical structure of the base pairs(B).

A                                                                                                B

Three of the base pairs in a row form what are known as codons. In firgure A above, you see G-C-A on strand one forming a codon. A series of codons form a gene. These three base pairs code for a particular amino acid. An amino acid is the basic building block of proteins. When you eat protein rich foods, it is the amino acids that your body is after so that they can be provided for the making of proteins that make up the various components of everything from cell walls, to your hair and fingernails. The amino acids are strung together in a process called translation involving messagner RNA(mRNA) and later transfer RNA(tRNA)to form long polypeptide chains, which fold into the proteins coded for by DNA. Here is a beautiful video that shows how the process works.

https://www.youtube.com/watch?v=gG7uCskUOrA

Scientists realized very early on in DNA research, that if they could modify individual genes they would be able to cure diseases, modify the operation of individual genes to produce desired effects, or even create synthetic life. This led to the birth of genetic engineering and the much maligned and misunderstood creation of genetically modified organisms or GMO’s. What is going on here is the modification of individual genes to produce proteins that not only directly cause a desired result, but also the turning of genes on and off through the interaction of proteins that are responsible for regulating individual genes. It is here that the devil in the details begins to rear its head.

I have given you just the bear minimum for understanding how DNA works to produce proteins. However the expression of a gene for a certain trait like eye color or a certain function like the production of insulin involves extremely delicate and complicate processes. Producing a genetic outcome is like producing a delicate dish from a complicated french recipe. In fact, DNA is more of a complex recipe book than a template or a blueprint. Recent research has proven that gene expression can be controlled by environmental or external factors. This is called epigenetics. So not only are there regulatory proteins that serve to regulate the production of other proteins, there are chemicals from the cellular environment that can influence gene expression and these chemicals can come from the foods you eat or the air that you breathe.

Basic research in molecular biology ultimately led to the discovery of enzymes(proteins) involved in the complicated process of gene expression. Some of these enzymes were responsible for the repair of damaged DNA. Damaged sections of DNA could be cut out and replaced with the correct sequence. In genetic engineering, researchers use these enzymes to cut out sections of DNA and replace the sections with other genes. For example, there are gene therapy techniques that treat certain forms of diabetes by replacing defective genes responsible for producing insulin with new insulin producing genes. This type of gene therapy can often lead to other damage to nearby genes and the process is not as specific as researchers would like. Quite by accident, researchers discovered a much more specific tool for excising unwanted genes and replacing them with “healthy” genes. This tool, is the CRISPR-cas9 tool. The following is an analogy that may help you understand the process.

CRISPR Analogy

Plagued for months by a home invader that she cannot see, Mary is fed up. The invader is invisible to the naked eye and enters at will systematically destroying parts of her home. She goes to a home security company to find a solution. She tells the salesman she wants a system that can detect and destroy the invader. The salesman comes up with the following solution.

He tells Mary he has a system that can make an image of the tiny invader AND that the imaging system, in combination with another system designed specifically to work with the image capture system, can deactivate or destroy not only the invader she’s after but any future invader that looks the same. The system, the salesman tells her is a kind of home immunity system. It will make her home immune to all future attacks.

Mary is thrilled but wants to know more. She asks the salesman for more details. He tells her that when the invader comes in there are tiny sensors always scanning for something that shouldn’t be there. When it detects a foreign invader an image recorder turns on and records the image of the invader. It will make many copies of the invader and spread them throughout her home, in combination with a small weapon designed only to eliminate an invader matching the image copied from the original invader. That way, he tells her, she is always protected. The details, he tells her are proprietary but he assures her the system will work flawlessly. Mary tells him, that’s okay. That’s all she needs to know.

As Mary is about to leave when suddenly she has a thought and turns to the salesman and explains that the world is a pretty nasty place with all sorts of bad guys and invaders. She wonders if the system can be adapted to use an image of someone or something already known to be destructive or bad, and then combine that image with the deactivation system and preemptively act to prevent the bad guy from acting.

The salesman smiles and tells Mary that she is very bright and insightful. That is exactly what our next generation of security systems will be designed to do, he explains. The salesman, excited by Mary’s interest and insight, begins to tell her all about how his company is developing a way to adapt the system to positive situations by creating a synthetic image , combining it with the deactivation system now modified not to destroy the original image but use that image to create a positive outcome.

Mary looks puzzled… Sensing that Mary doesn’t quite get it the salesman comes up with an example. He explains, “I can see you are a little confused by all this. Let me explain further. Suppose you have a faulty plumbing system in your house, but we know what a healthy working plumbing systems looks like. We can create an image of the healthy system, combine it with our modification system, send it to your house and it will recognize the specific plumbing problem, remove it and replace it with a working system. The reason this works is because the plumbing problem and the healthy plumbing system look almost identical, except for the broken part causing the problem.”

“Wow”, Mary responds. That would be awesome. When will that be available? It’s already in the works” the salesman responds. “That will be totally cool,” Mary says. She waves goodbye and is off with her new security system to give her house immunity from the home invader.

The above story is an analogy for how the CRISPR-cas immune system works in a bacteria. Mary’s home is analogous to a prokaryotic cell. The tiny invader is viral DNA, the security imaging system is the cellular process of cutting up and then copying segments of the viral DNA and putting them into what is known as the CRISPR RNA complex analogous to the combination system the home security guy sells to Mary. In the cell this complex is made up of the viral RNA segments(copied from the original viral DNA) combined with a protein. The complex is capable of recognizing invading viruses that look the same as the copied virus and deactivating or destroying any future invaders or viruses. Here is a diagram of the cellular CRISPR system that may help you visualize how this works. The diagram should be read clockwise beginning with double stranded viral DNA, which circles back in a feedback loop to recognize other double stranded viral DNA. The colored numbers refer to the analogy summarized below.
DSC_6498

E is analogous to the entrance to Mary’s house
#1 In the diagram is analogous to the tiny home invader in Mary’s house
#2a, b, c Are analogous to the imaging system the salesman gives Mary that makes copies of the tiny invader
#3 is analogous to the deactivation system that combines with the copies of the tiny invader
#4 Is analogous to the process of targeting and deactivating any future tiny invaders

 

CRISPR is the abbreviation for, Clustered regularly-interspaced short palindromic repeats. In prokaryotic cells(such as bacterial cell which do not have a nucleus) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of “spacer DNA” from previous exposures to a bacterial virus or plasmid As shown in the previous figure above.

So what is going on here? In a nutshell, a virus infects a bacterium with its DNA. In defense, the bacterium binds a protein, cas, to the viral DNA, which chops up the viral DNA into pieces. Some of these pieces are used as “spacers” within the bacterium’s CRISPR array. The bacterium then copies or transcribes the spacer pieces into a strand(this stand is a copy of part of the viral DNA that originally infected the bacterium). This strand is then processed or transribed into segments of RNA called CRISPR RNA, crRNA which then combines with another protein called CASIII to form a complex. This complex is programed to recognize the original viral DNA. Upon recoginizing the viral DNA the CAS crRNA complex attacks and inactivates the viral DNA. You can think of it as a kind of negative feedback loop. Once the bacterium “knows” what the invading virus looks like, it is then equipped to deactivate it.

The most important aspect of this process for general gene editing is the fact that the CASIII crRNA complex recognizes the specific sequence on the viral DNA as a result of having taken segments of viral DNA during the initial infection. So now the question is, how do researchers use this information to produce gene editing tools.

Let’s say instead of viral DNA containing information that allows it to infect a bacterium and reproduce within it, thus destroying the bacteria, the researcher constructs a carrier virus that has a synthetically produced DNA segment which represents say the gene for producing insulin. The gene segment for insulin then becomes the “spacer DNA” which is ultimately used in the crRNA Cas complex, which recognizes that specific segment of DNA. But this time instead of recognizing it within a virus, it recognizes it in a human cell within the pancreas. It will specifically target that segment ONLY and either excise it (cut it out) and or replace it depending on how the researcher has programed the crRNA Cas complex.

However, here is the rub and hence the controversy. As some of you forward thinkers out there may have already guessed, this is a powerful gene editing tool that can be used in many, many different ways. The most controversial is its use to modify the genes of a human embryo. You’ve probably already seen hints of this in the sensational, though very real, headlines about designer babies. As sensational as those headlines may be, they point to a very real ethical dilemma. The CRISPR gene editing tool would make possible for the first time the modification of a gene that could then be passed along to future generation. This also imparts the ability to manipulate our evolutionary destiny.

On the positive side, let us suppose that the gene for hemophilia(a sex-linked genetically inherited disease)could be modified in the male embryo that has hemophilia. Using CRISPR-cas9 the faulty gene could be removed and replaced with a healthy gene. The male born from that embryo would be cured of hemophilia AND would pass on the healthy gene to his progeny. This and other gene replacements hold great promise for curing diseases and preventing genetic diseases from being passed on to future generations. But what if you wanted to modify or add genes that could give the recipient “super powers”, like enhanced intelligence, increased athletic ability, a guaranteed set of physical features? How far would we be from creating Star Trek’s, Kahn and his army of super-humans?

I made the subtle point early on that the devil in gene editing is very much in the details. Genes very often have multiple functions. They may code for a particular trait like eye color or a specific functions like insulin production, but those same genes may play a role in the regulation of other genes. What we don’t know about the workings of the human genome far outweighs what we do know. While we have celebrated the mapping of the entire human genome, how the various genes interact with one another is only beginning to be understood. Then, there is the new discovery of epigenetics mentioned earlier, where environmental factors play a role in gene expression. Many genes lie dormant for nearly a lifetime until turned on by some environmental factor or through normal regulatory processes. There is a belief by some biologists that there are many genes that are “dark” meaning they have no function. I believe this is a matter of our lack of full knowledge of the workings of genetic systems. Many of these so called dark genes may prove to be very much in the light and have important functions.

This is the reason that gene editing, especially embryonic gene editing is so fraught with controversy and ethical problems. There are surely to be many unintended consequences as this research and well meaning genetic therapies are applied. It is these and other issues that The International Summit on Gene Editing tackled in a meeting last December. At the conclusion of the summit a statement was issued summarizing the recommendations, which resulted from the discussions between researchers, ethicists, clinicians and public interests groups. The statement concluded with,

“While each nation ultimately has the authority to regulate activities under its jurisdiction, the human genome is shared among all nations. The international community should strive to establish norms concerning acceptable uses of human germline editing and to harmonize regulations, in order to discourage unacceptable activities while advancing human health and welfare.

Since this statement was made, CRISPR research has continued at Gangzhou University in China and at the Francis Crick Institute in London by developmental biologist Kathy Niakan. A research paper by the Chinese researchers reports attempts to modify the gene that codes for the CCR5 protein receptor on T cells. It is this receptor that the HIV uses to attach to and infect T cells. Modifying the gene to produce a variant of CCR5 could prevent HIV from infecting people and giving future generations a kind of natural immunity to HIV. The controversy the paper caused is rooted in what another researcher described as, “. . .the science is going forward before there’s been the general consensus after deliberation that such an approach is medically warranted,”

There are currently many crisis in the world, from ISIS to the European migrant problem; from Libya to Syria; Afghanistan to the North Korea and on and on. These crisis loom large and cast a huge shadow over humanity, often blocking the light that would expose a more insidious crisis. CRiSPR-cas 9 bio-technology could change the evolutionary direction of humanity toward an unknown destination full of unparalleled risks. The scientific community is to be commended for convening the International Summit on Gene Editing. However, a major element was missing from that summit, and that is participation by ordinary citizens through science based civic engagement. I am the first to admit that such civic engagement assumes a citizenry educated enough to make thoughtful and meaningful contributions to the dialog without resorting to conspiracy theory and hyperbole.

It is my hope that this brief article will begin to equip the broader populace with the tools necessary to understand these complex scientific and ethical problems posed by some of the greatest scientific achievements in the history of humankind; and that once so equipped scientists and citizens will together be able to manage both the promise and the peril of CRISPR-cas9

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2 replies »

  1. It is a very well written and informative article, Don. I can see that it poses an ethical dilemma requiring much thought. I miss your science classes as this would lend itself to a most interesting and lively discussion. Maybe you’ll consider just one more??????

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