AP Biology class blog for discussing current research in Biology

Tag: Cas9

CRISPR Mini | New Territory Unlocked

For over a million years, DNA has centered itself as the building block of life. On one hand, DNA (and the genes DNA makes up) shapes organisms with regard to physical appearance or ways one perceives the world through such senses as vision. However, DNA may also prove problematic, causing sickness/disease either through inherited traits or mutations. For many years, scientists have focused on remedies that indirectly target these harmful mutations. For example, a mutation that causes cancer may be treated through chemotherapy or radiation, where both good and bad cells are killed to stop unchecked cell replication. However, a new area of research, CRISPR, approaches such problems with a new perspective.

The treatment CRISPR arose to answer the question: what if scientists could edit DNA? This technology involves two key components – a guide RNA and a CAS9 protein. Scientists design a guide RNA that locates a specific target area on a strand of DNA. This guide RNA is attached to a CAS9 protein, a molecular scissor that removes the desired DNA nucleotides upon locating them. Thus, this method unlocks the door to edit and replace sequences in DNA and, subsequently, the ways such coding physically manifests itself. Moreover, researchers at Stanford University believe they have further broadened CRISPR’s horizon with their discovery of a way to engineer a smaller and more accessible CRISPR technology.

This study aimed to fix one of CRISPR’s major flaws – it is too large to function in smaller cells, tissues, and organisms. Specifically, the focus of the study was finding a smaller Cas protein that was still effective in mammalian cells. The CRISPR system generally uses a Cas9 protein, which is made of 1000-1500 amino acids. However, researchers experimented with a Cas12f protein which contained only 400-700 amino acids. Here, the new CasMINI only had 529 amino acids. Still, the researchers needed to figure out if this simple protein, which had only existed in Archaea, could be effective in mammals that had more complicated DNA.

To determine whether Cas12f could function in mammals, researchers located mutations in the protein that seemed promising for CRISPR. The goal was for a variant to activate a protein in a cell, turning it green, as this signaled a working variant. After heavy bioengineering, almost all the cells turned green under a microscope. Thus, put together with a guide RNA, CasMINI has been found to work in lab experiments with editing human cells. Indeed, the system was effective throughout the vast majority of tests. While there are still pushes to shrink the mini CRISPR further through a focus on creating a smaller guide RNA, this new technology has already opened the door to a variety of opportunities. I am hopeful that this new system will better the general well-being as a widespread cure to sickness and disease. Though CRISPR, and especially its mini version, are new tools in need of much experimentation, their early findings hint at a future where humans can pave a new path forward in science.

What do you think? Does this small CRISPR technology unlock a new realm of possibility or does it merely shed light on scientists’ lack of control over the world around us?

Unnatural Selection: The Future of The Future?

Imagine it’s Saturday night, you are snowed in until the morning and you need a way to pass the time. Like many people, you resort to Netflix. Upon browsing through the vast selection of horror, comedy, and romantic films, you decide you are in the mood for a documentary. Scrolling through the options, you stop at a title that grabs your attention: Unnatural Selection.

Since you are an AP Biology student, you immediately connect the words “Natural Selection” to the work of Charles Darwin, the study of genetics, and most importantly: evolution. In brief, natural selection is the survival and reproduction of the fittest, the idea that organisms with traits better suited to living in a specific environment will survive to reproduce offspring with similar traits. Those with unfavorable traits may not be able to reproduce, and therefore those traits are no longer passed down through that species. Natural selection is a mechanism for genetic diversity in evolution, and it is how species adapt to certain environments over many generations.

If genetic diversity enables natural selection, then what enables unnatural selection? Well, If natural selection eradicates unfavorable traits naturally, then unnatural selection essentially eradicates unfavorable traits or promotes favorable traits artificially.

The Netflix docuseries “Unnatural Selection” focuses on the emergence of a new gene-editing technology named CRISPR (an acronym for “Clustered regularly interspaced short palindromic repeats”). CRISPR is a revolutionary new method of DNA editing, which could help cure both patients with genetic diseases and patients who are at risk of inheriting unwanted genetic diseases. The two pioneers of this technology, Emmanuelle Charpentier and Jennifer Doudna, recently won Nobel Prizes in Chemistry for their work on CRISPR.

CRISPR illustration gif animation 1

Animation of CRISPR using guide RNA to identify where to cut the DNA, and cutting the DNA using the Cas9 enzyme

CRISPR works with the Cas9 enzyme to locate and cut a specific segment of DNA. Scientists first identify the sequence of the human genome, and locates a specific region that needs to be altered. Using that sequence, they design a guide RNA strand that will help the Cas9 enzyme, otherwise known as the “molecular scissors” to locate the specific gene, and then make precision cuts. With the affected region removed, scientists can now insert a correct sequence in its place.

Using the bacterial quirk that is CRISPR, scientists have essentially given anyone with a micropipette and an internet connection the power to manipulate the genetic code of any living thing.

Megan Molteni / WIRED

CRISPR is just the beginning of gene editing, introducing a new field of potential gene editing research and applications. But with great power comes great responsibility — and great controversy. Aside from the obvious concerns, people speculating the safety, research, and trials of this new treatment, CRISPR headlines are dominated by a hefty ethical dilemma. On one hand, treating a patient for sickle cell anemia will rid them of pain and suffering, and allows their offspring to enjoy a normal life as well. However, by eliminating the passing down of this trait, sickle cell anemia is slowly eliminated from the human gene pool. Rather than natural selection choosing the path of human evolution — we are. We are selecting which traits we deem “abnormal” and are removing them scientifically. Although CRISPR treatment is intended to help people enjoy normal lives and have equally as happy children, putting evolution into the hands of those evolving can result in more drastic effects in the future.

For our generation, CRISPR seems like a magical cure for genetic diseases. But for future generations, CRISPR could very well be seen as the source of many problems such as overpopulation, low genetic diversity, and future alterations such as “designer babies.” Humans have reached the point where we are capable of our future. Is CRISPR going to solve all of our problems, or put an end to the diverse human race as we know it? Comment how you feel down in the comments.


Redesigned Cas9 protein provides safer gene editing than ever before!

Gene editing is a group of technologies that give scientists the ability to change an organism’s DNA. These technologies allow genetic material to be added, removed, or altered at particular locations in the genome. Several approaches to genome editing have been developed. A recent one is known as CRISPR-Cas9, which is short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9. The CRISPR-Cas9 system has generated a lot of excitement in the scientific community because it is faster, cheaper, more accurate, and more efficient than other existing genome editing methods.

One of the challenges that come using CRISPR-based gene editing within humans is that the molecular machinery may sometimes make edits to the wrong section of a host’s genome. This is problematic because it creates the possibility that an attempt to repair a genetic mutation in one location in the genome could accidentally create a dangerous new mutation in another spot. Scientists at The University of Texas at Austin have redesigned a key component of a widely used CRISPR-based gene-editing tool, called Cas9, to be thousands of times less likely to target the wrong stretch of DNA while remaining just as efficient as the original version, making it potentially much safer.

The CRISPR-Cas9 system works similarly in the lab. Researchers create a small piece of RNA with a short ‘guide’ sequence that binds to a specific target sequence of DNA in a genome. The RNA also binds to the Cas9 enzyme. As in bacteria, the modified RNA is used to recognize the DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location. Although Cas9 is the enzyme that is used most often, other enzymes can also be used. Once the DNA is cut, researchers use the cell’s own DNA repair machinery to add or delete pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customized DNA sequence.

Other labs have redesigned Cas9 to reduce off-target interactions, but so far, all these versions improve accuracy by sacrificing speed. SuperFi-Cas9, as this new version has been named, is 4,000 times less likely to cut off-target sites but just as fast as naturally occurring Cas9. Scientists say you can think of the different lab-generated versions of Cas9 as different models of self-driving cars. Most models are really safe, but they have a top speed of 10 miles per hour.

In my opinion, setting aside any and all ethical concerns, genome editing is of great interest in the prevention and treatment of human diseases. Currently, most research on genome editing is done to understand diseases using cells and animal models. Scientists are still working to determine whether this approach is safe and effective for use in people. It is being explored in research on a wide variety of diseases, including single-gene disorders such as cystic fibrosis, hemophilia, and sickle cell disease. It also holds promise for the treatment and prevention of more complex diseases, such as cancer, heart disease, mental illness, and human immunodeficiency virus (HIV) infection.

Is CRISPR the COVID-19 Cure?

New Developments In CRISPR Gene Editing Technology Show Promising Advances In Possible COVID-19 Antiviral Pill

CRISPR Gene Editing. If you have never heard of it, don’t worry, I hadn’t either. When google searching CRISPR Gene editing, I went straight to Wikipedia for the simple answer that it is a procedure done in molecular biology, in which the genomes of a living organism can be modified with extremely high precision. One of its many applications is the treating and prevention of disease, enabling researchers to edit DNA and use the natural defense system of bacteria to target and destroy the genetic material of viruses. In a new study from this summer, Dr. Sharon Lewin and her team of researchers at the Peter Doherty Institute for Infection and Immunity at the University of Melbourne believe they may have harnessed CRISPR’s gene editing abilities to block the replication of COVID-19. 

Very similar to the replication of DNA, RNA replication begins with a single strand of “Template” RNA. In DNA, because it can only be replicated in one direction (5’-3′), and the strands run antiparallel, each strand is built in opposite directions creating one leading strand and one lagging strand. However, RNA only needs one strand made because it is single-stranded instead of a double. In SARS-CoV-2, an enzyme called RNA-Dependent RNA Polymerase adds nucleotides in the 5’-3′ direction, replicating the template RNA. Because humans have DNA, we don’t copy RNA; instead, we transcribe it to make proteins. Therefore this RNA replication process does not occur in humans and only in viruses.

Lewins’ team designed the gene editing to target single strands of RNA, like those found in COVID-19. CRISPR is most commonly associated with Cas9, an RNA-guided enzyme that cleaves foreign nucleic acids. However, Lewin and her team used a different enzyme, Cas13b, which could cleave RNA instead. Targeting specific sites on the RNA strands of SARS-CoV-2, Cas13b binds to the RNA and destroys the part of the virus needed to replicate, “Once the virus is recognized, the CRISPR enzyme is activated and chops up the virus,” said Lewin. She continues to explain that although the COVID-19 vaccines are highly effective, there is still a clear and urgent need for treatment once the disease is contracted. The ideal treatment would be an antiviral drug that could be taken shortly after the patients tested positive for COVID-19, “That’s what we hope to achieve one day with this gene scissors approach.” 

CRISPR Cas9 technology

Having written in previous blog posts about my mother’s struggles with COVID-19, my dad also had a very different yet real struggle. Like most people, my dad, having somehow not contracted COVID from my mom at the beginning of quarantine, was very fearful of getting sick himself. Fortunately, my dad has still never had COVID (knock on wood). This is great because he has remained healthy; however, it also had downsides. For my brother and me, being both kids and relatively healthy, when we contracted COVID in mid-August, it was nothing more than a rough cold. A cold that, after ten days, not only was gone but enabled me to feel some sense of temporary immunity to the virus and allowed me to feel comfortable going out with friends and returning to some level of normalcy. My dad never got this. Because he never contracted COVID, he lived a completely secluded life until this past February (when he gave up and began going out in public). If my family and I went to a mall, he would wait in the car. If we ate out, he would wear a mask the whole time and not eat until we got home. The fear for my dad was not specifically getting covid but not having some antiviral drug to take once he contracted the virus. A solution like Dr. Lewins would have been and still would be a life-changer for many families who still live in fear of getting sick from COVID-19.  

Although this breakthrough in RNA CRISPR technology is remarkable, the study was performed in lab dishes and is still waiting for testing on animals or humans. Additionally, CRISPR technology medicines have not been approved to treat any diseases. Unfortunately, we are probably a couple of years away from a widely available treatment. 

Could Christmas Island rats make a comeback? Thanks to CRISPR gene editing, they might!

From climate change to overhunting by humans, there are many factors which contribute to the extinction of species in the animal kingdom. The Christmas Island rat, also known as Maclear’s rat, went extinct a century ago in what is believed to be the first and only case of extinction of a species due to disease. It has always been believed that once a species goes extinct, it is gone for good. That is until recently when scientists began experimenting with “de-extinction” efforts to bring back the Christmas Island rat.

As published March 9 in the science journal, Current Biologya team of paleo geneticists from the University of Copenhagen recently conducted a study into gene sequencing the Christmas Island rat, in order to estimate the possibilities of future gene editing experiments which could bring the species “back to life”. The process of genetic editing for de-extinction efforts, as explained by the research team in their abstract, consists of first identifying the genome of the species and then editing the genes of similar species to make it more similar to that of that extinct one. The team used frozen somatic cells of the extinct rats, cells with a 2n number of chromosomes which are made during the process of mitosis. The team was able to sequence the rats’ genome, aside from some small portions which remain missing. They then had to identify the modern species which they could gene edit. Their findings established that the Christmas Island rat shares around 95% of DNA with the modern Norway brown rat. At this point, it

Now that the rat’s genome has been sequenced to the best of the team’s ability and a similar species has been identified, the gene editing possibilities are endless, especially with CRISPR technologies and techniques. “CRISPR” stands for Clustered Regularly Interspaced Short Palindromic Repeats in DNA sequencing. This system was discovered by a group of scientists, led by Dr. Emmanuelle Charpentier. CRISPR uses Cas9, an enzyme which cuts DNA at specified sections as guided by RNA. There are three different types of edits drone with CRISPR technology: disruption, deletion, or correction/insertion. Disruption editing is when the DNA is cut at one point and base pairs are either added or removed to inactivate a gene. Deletion editing is when the DNA is cut at two points and a larger sequence of pairs is removed. Correction/insertion editing is when a new gene is added into a sequence using homology directed repair.

Thomas Gilbert, the lead scientist on the team, says that he would like to conduct CRISPR gene editing experiments on living species of rats before attempting to replicate the DNA of an extinct species. For example, attempting to mutate the DNA of the Norway brown rat into that of the common black rat. Once this experiment is conducted, the possibilities of reviving the Christmas Island rat will be more clear. Until then, we can only hope! Do you think it’s possible to see the Christmas Island rat revived anytime soon?

Genetically Modified Babies?

A decade or two ago, the idea of being able to modify embryos was straight out of a science-fiction movie. However, last November, Chinese scientist He Jiankui genetically modified twin girls’ embryos to have resistance to the HIV virus using a process called CRISPR. His actions have sparked a global panic, as many people feel that current regulations are not enough to keep the scientific community’s actions ethical.

To understand this issue, it is important to understand its individual components. CRISPR is a gene-editing tool that was discovered in 2007 and became widely used in 2013. Essentially, a scientist decides what portion of DNA they would like to alter, and transcribes the sequence into RNA. This RNA finds the portion of DNA with the specific code and then the Cas9 enzyme “cuts” the DNA, allowing a new sequence of DNA to take its place.

The image depicts functions of CRISPR Cas9 technology.

Dr. He used CRISPR Cas9 technology to try to block the HIV pathways in twin girls while they were still embryos. As this experiment was recent, the long-term effects of it are unclear. In addition, as these girls were not developed at the time of their gene editing, they did not give consent to have a treatment that could be detrimental to their health. Furthermore, looking at the Centers for Disease Control website, HIV is primarily acquired by the use of unsafe needles to inject drugs and sexual contact. Using clean needles and condoms can greatly decrease one’s risk of getting HIV, and if a HIV-positive person takes suppression medicines, the viral content of HIV in their blood can become undetectable. Dr. He’s actions gave the twin girls undue risk, with little possible benefit.

In the future, this method of gene editing may be used to prevent or treat genetic diseases, but people have little knowledge of the long-term implications of using this technology on embryos. At the moment, the lack of global legislation regarding this gene-editing technology leaves a lot to be wondered about the future of this tool. According to Victor Dzau who works in the United States National Academy of Medicine, “The silver lining is that the world was awakened by the conduct of Dr. He, and we are all working very, very hard with all good intentions to make sure that this doesn’t happen again—not in the fashion that He did it. And that someday, if and when the technology is ready—and I think all of us are very bullish about this technology—that it will be helping humankind in the right way, knowing the risks and knowing the benefits.” After Dr. He’s experiment, many are in favor of halting the use of CRISPR on human embryos for at least five more years, so more research can be done on the subject. However, legislation, which the world has seen little of, holds a stronger weight than mere recommendations. In Russia, Denis Rebrikov is planning to create CRISPR babies, and regulations in the country regarding his specific goals remain unclear. How will CRISPR embryo editing evolve in the coming decades? Will CRISPR gene editing be as common someday as IVF is today?


New anti-CRISPR Proteins Serving as Impediments to this Miraculous System.

CRISPR-Cas9 systems are bacterial immune systems that specifically target genomic sequences that in turn can enable the bacterium to fight off infecting phages. CRISPR stands for “clusters of regularly interspaced short palindromic repeats” and was  first demonstrated experimentally by Rodolphe Barrangou and a team of researchers at Danisco. Cas9 is a protein enzyme that is capable of cutting strands of DNA, associated with the specialized stretches of CRISPR DNA.

Diagram of the CRISPR prokaryotic antiviral defense mechanism.

Recently, a blockage to the systems was found by researchers which are essentially anti-CRISPR proteins. Before, research on these proteins had only showed that they can be used to reduce errors in certain genome editing. But now, according to Ruben Vazquez Uribe, Postdoc at the Novo Nordisk Foundation Center for Biosustainability (DTU), “We used a different approach that focused on anti-CRISPR functional activity rather than DNA sequence similarity. This approach enabled us to find anti-CRISPRs in bacteria that can’t necessarily be cultured or infected with phages. And the results are really exciting.” These genes were able to be discovered by DNA from four human faecal samples, two soil samples, one cow faecal sample and one pig faecal sample into a bacterial sample. In doing so, cells with anti-CRISPR genes would become resistant to an antibiotic while those without it would simply die. Further studies found 11 DNA fragments that stood against Cas9 and through this, researchers were ultimately able to identify 4 new anti-CRIPRS that “are present in bacteria found in multiple environments, for instance in bacteria living in insects’ gut, seawater and food,”  with each having different traits and properties.  “Today, most researchers using CRISPR-Cas9 have difficulties controlling the system and off-target activity. Therefore, anti-CRISPR systems are very important, because you want to be able to turn your system on and off to test the activity. Therefore, these new proteins could become very useful,” says Morten Sommer, Scientific Director and Professor at the Novo Nordisk Foundation Center for Biosustainability (DTU). Only time will tell what new, cool, and exciting discoveries will be made concerning this groundbreaking system! What else have you guys heard? Comment below!

Cas9: Dormant Killers?

Woah. Pretty aggressive title, but it did grab your attention, didn’t it?

What the Heck Even Is Cas9?

Well, Cas9 is an integral part of the CRISPR-Cas9 system. The Cas9 is able to latch on to a piece of DNA (it grabs on based on a piece of guide RNA, or gRNA in the complex) and cuts it. When this happens, the cell is like, “Oh no! Broken DNA! We must fix!”.  Now I know what you’re thinking… why fix something that ain’t broke? Well, what happens is that (in science being done today) the piece of DNA already has something wrong with it that the cell isn’t aware of such as a point mutation. Breaking the DNA just brings the problem to the cell’s attention so that it can fix the DNA. This was a defense mechanism in bacteria to work against viruses and the like by ‘cataloging’ the viruses they’ve encountered and using the system to cut up the viral DNA, but scientists thought it was pretty cool so they decided to figure out a way to make it work in animals.

Alright… Why Are They Killers?

Well… what these scientists have done is that they have developed a way to basically use the CRISPR-Cas9 system as a defense mechanism for diseases prevalent in humans and plants such as cancer or the West Nile virus. Basically, how this would work is that at the end of the Cas9 researches added a little protein tail that could only be cut by a specific protein. When this protein is cut, then the Cas9 would be ‘activated’ and would do its defensive duties. Scientists could change this protein to target different things, such as adding a protein that cancer cells make enzymes for. It was also used in plants to target certain viruses. So in short, this Cas9 DNA destruction mechanism is dormant until presented with the foe it has been essentially trained to fight.


So Is The World Cancer-Free?

Unfortunately not, my friend. Even though this is revolutionary stuff happening, the research done is mostly for plants in order to resist infectious diseases and viruses. It has, however, offered us insight into how we can manipulate this system to serve us in a beneficial way. They also, during this whole protein-tail process, figured out that they can get the protein to still work in different configurations that make it easier to add certain protein tails. They determined that by cutting Cas9’s amino acid chain and rearranging it in certain ways, it can have easier protease recognition sites as well as attachment sites for the protein.

Do you think this system will revolutionize medicine?

Will this eventually replace vaccines?

Will the continued research into gene editing come back to haunt us in the end?

What does the future hold for CRISPR-Cas9?

Genome editing, or the technologies in which scientists can change the DNA of an organism, is on the rise, especially with its latest development, CRISPR-Cas9, the most efficient method of all of the methods to edit DNA.

Like many other discoveries in science, CRISPR-Cas9 was discovered through nature. Scientists learned that certain bacteria capture snippets of DNA from invading viruses, making DNA segments called CRISPR arrays, helping them remember the virus to prepare for future invasions of that virus. When they are confronted with that virus again, RNA segments from the CRISPR arrays are created which target the DNA of the virus, causing the enzyme Cas9 to cut the virus’ DNA apart, which would destroy the virus.


We use the same method in genome editing with CRISPR-Cas9 by creating RNA that binds to a specific sequence in a DNA strand and the Cas9, causing the Cas9 to cut the DNA at that specific sequence. Once this is done, the scientists create a sequence to replace the one that was cut to get the desired genome.

This technology is most prominently used to attempt to treat diseases, where the somatic cells’ genomes are altered which affect tissues, as well as prevent genetic diseases where the sperm or egg’s genome is changed. However, the latter causes some serious ethical concerns of whether we should use this technology to enhance human traits. But this begs the question that if we start using it more and more to prevent genetic diseases, will this open the door for it to be used in new ways?

Cas9: A Clue Into Making Gene Editing Safer

CRISPR is a revolutionary system that edits the DNA of living organisms with ease. The gene-editing technology offers scientists insight into genetic diseases and is widely used in biotech and agriculture, as well as to treat cancer and viral infections. But the CRISPR system and its mechanisms are not yet fully understood. However, researchers at the Ohio State University have reported that they have figured out the mechanism of how the CRISPR system figures out where and when to cut the DNA strands. This is particularly revolutionary as it provides insight into preventing gene-cutting errors.

Cas9 is an enzyme that is used by the system to target and cut out or insert specific genes. In the second of two paper published in the Journal of the American Chemical Society, the team invalidates the widely-held belief that the enzyme cuts DNA evenly. Professor of chemistry and biochemistry Zucai Suo explains that instead of cutting both sides of the DNA double-helix to the same length, Cas9  actually trims each side to uneven lengths. Ohio State doctoral student Austin Raper and his co-authors determined that the two different parts of the “Cas9 molecule communicate with each other to set the location and timing of a cut”. The first part of the molecule sets forth to cut its respective DNA strand and changes shape and signals to the second part to cut its respective second strand.

Crystal Structure of Cas9 Enzyme

Suo says that he hopes their work allows for scientists to minimize and eventually eliminate gene-editing errors. CRISPR rarely target unintended genes, but gene-editing errors can have very serious consequences. For example, if the system accidentally cut a tumor suppressor gene from a person’s DNA, they would be much more likely to develop cancer. As Raper says, it is important to understand CRISPR and the Cas9 enzyme mechanisms in order to allow CRISPR to advance to its full potential.



The Future of CRISPR

CRISPR is starting to become more and more of a reality as Harvard professor David Liu continues to work on it. Liu was the person who originally developed CRISPR first base editor which allowed for single letter changes in the genetic code. Liu has come up with two new features to CRISPR-Cas9.

The first is called cellular detective or CAMERA(CRISPR-mediated analog multievent recording apparatus systems). What this function does is it finds the genetic problem that is responsible for the disease someone is experiencing. Cas9 will record all the cell data and piece info together, which overall will provide more information about cancer, stem cells, aging, and overall disease.

Photo Source

The second finding is referred to as sharp scissors which is a CRISPR enzyme. Sharp scissors are way more precise and accurate than the old enzyme making is much safer. The scissors depend on specific DNA to find the region where it is supposed to cut or edit. CRISPR is progressing and as more research is being done could be used on humans in the future.


CRISPR/Cas9: Is it really the cure?

There are many benefits to the CRISPR/Cas9 defense system. But, do the pros outweigh the cons?

CRISPR is a molecule that can be programmed to target a specific sequence in a genome. It guides an enzyme, such as Cas9, to chop the code like scissors. There have been many studies and tests done using the defense. The most important advantages of CRISPR/Cas9 over other genome editing systems are its simplicity and efficiency. Since it can be applied directly in the embryo, CRISPR/Cas9 reduces the time required to change certain genes compared to other systems.

However, many attempts to use this mechanism have failed. Using the mechanism is not as easy as it sounds. A Cas9 repair is not always precise. On ZMEScience, one HIV patient tried the process. But, the HIV cells were only made stronger. Researchers equipped T-cells to hurt the virus with the enzyme Cas9. T cells are a type of lymphocyte that play a central role in cell-mediated immunity. T cells equipped with Cas9 were seen to successfully hurt the HIV genome, and make it unable to properly reproduce. This project led by Chen Liang from McGill University in Montreal, Canada seemed to work fine. But, the team noticed that two weeks later T cells were producing copies of virus particles that had escaped the CRISPR attack. The area around Cas9 only developed more mutations, aka it made the HIV stronger. It is also impossible to direct the Cas9 exactly where one wants it to go. So in essence, it is a risky gamble.

Although there are hopes for this technique to be more refined and successful in the future, for now, its uses are limited.

For more information click here.

What is CRISPR-Cas9?

CRISPR-Cas9 is a new(ish) technology that is used for knocking out human genes in cell lines for the purpose of seeing what these genes do. CRISPR-Cas9 has a “protein scissor”, the cas-9 protein, and a location that shows the cas9 where to bind to. The “location” is actually a strand of RNA that is complementary to a specific strand of DNA. This RNA strand is like glue in that it binds to the DNA and allows the Cas9 to cut the DNA. This process or the CRISPR-Cas9 technology is like an endless cycle of cutting and repairing DNA until the repair enzyme can no longer repair the DNA or makes a mistake. This technology can make the process of cutting and disabling genes five times faster. It allows scientists to edit parts of a genome by altering, removing, or adding certain sections of DNA. While this technology can be very useful in trying to understand what genes do it does have a downside, “these approaches are costly and time-consuming to engineer, limiting their widespread use, particularly for large scale, high-throughput studies.” The picture below shows what this process looks like on a very basic scale. Hopefully this technology will eventually allow us to fully understand what every gene does.


The Miracle of CRISPR/Cas9 in Gene Editing

Some scientists say, “you can do anything with CRISPR” and others are absolutely astonished and amazed.

CRISPR can rapidly change any gene in any animal or plant with ease. It can fix genetic diseases, fight viruses, sterilize mosquitos and prepare organs for transplant. The possibilities are endless – and the prospect of designer babies isn’t far off.

Dead Cas9 can fix a single base pair typo in DNA’s genetic instructions. It can convert a C-G into a T-A pair. Also, we can attach fluorescent tags to dead Cas9 so researchers can locate and observe DNA or RNA in a living cell. Dead Cas9 can also block RNA Polymerase from turning on a gene, in CRISPRi. In CRISPRa, a protein that turns on genes is fused to dead Cas9.

CRISPR can be used for anything involving cutting DNA. It guides molecular scissors (Cas9 enzyme) to a target section of DNA & works to disable or repair a gene, or insert something new.

Many scientists have been thinking of improvements for this miracle gene editor. RNA Biologist Gene Yeo compares the original Cas9 to a Swiss army knife with only one application – a knife. He says that by bolting other proteins and chemicals to the blade, they transformed the knife into a multifunctional tools.

CRISPR/Cas9 is special because of its precision. It is much easier to manipulate and use compared to other enzymes that cut DNA. By using “guide RNA” it can home in on any place selected by the researcher by chemically pairing with DNA bases.

While Cas9 does have some problems, scientists definitely see the potential for greatness with a few tweaks. They wanted to ensure permanent single base pair changes, and they increased that from 15 to 75 percent. Liu used a hitchhiking enzyme called cytidine deaminase.

Scientists researched chemical tags on DNA called epigenetic marks. When scientists placed the epigenetic marks on some genes, activity shot up. This provided evidence that the mark boosts gene activity.

Case can also revolutionize RNA biology. The homing ability of CRISPR/Cas9 is what makes this seem possible. It was found that Cas9 could latch on to mRNA.

CRISPR/Cas9 was first found in bacteria as a basic immune system for fighting viruses. It zeroes in on and shreds the viral DNA. Half of bacteria have CRISPR immune systems, using enzymes beyond Cas9.

Overall scientists predict that in the next few years, results will be amazing. The many ways of using CRISPR will continue to multiply and we will see where science takes us.


Other Sources:

Anti-CRISPR Proteins: What are they and can they be beneficial?

NIH Image Gallery Image Link

Understanding CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

For many bacteria, one line of defense against viral infection is the RNA guided “immune system” known as CRISPR-Cas. This particular complex is unique because of its ability to recognize viral DNA and trigger its destruction. Scientists have used CRISPR to degrade sections of viral RNA and use the CRISPR systems to remove unwanted genes from an organism. CRISPR proteins have also been studied with the hope of eliminating serious disease and illnesses. However, this CRISPR system does not always work do to anti-CRSPR proteins that inhibit the complex from working properly.


According to an article on ScienceDaily, researchers have finally discovered how these anti-CRISPR proteins work! Research done by biologist Gabriel C. Lander from the Scripps Research Institute, discovered that anti-CRISPR proteins work by inhibiting CRISPR’s ability to identify and attack viral genomes. Just like there are different CRISPR systems, there are multiple anti-CRISPR proteins as well. One in particular mimics DNA to throw the CRISPR-guided detection machine off its course. Scientists have been able to further discover certain aspects of CRISPR and anti-CRISPR systems by using a high-resolution imaging technique called cryo-electron microscopy. They have discovered that the CRISPR surveillance complex analyzes a virus’s genetic material to see where it should attack by having proteins within the complex wrap around the CRISPR RNA, exposing specific sections of bacterial RNA. These sections of RNA then scan viral DNA, looking for genetic sequences they recognize. Lander describes these proteins as being very clever because they “have evolved to target a crucial piece of the CRISPR machinery. If bacteria were to mutate this machinery to avoid viral attacks, the CRISPR system would cease to function.” Therefore, CRISPR systems cannot avoid anti-CRISPR proteins without completely chancing the mechanism used to recognize DNA. Another type anti-CRISPR protein works a bit differently. Based on its location and negative charge, this anti-CRISPR protein acts as a DNA mimic, fooling CRISPR into binding this immobilizing protein, rather than an invading viral DNA.

Can Anti-CRISPR Proteins be beneficial?

Researchers are saying that the understanding of how these anti-CRISPR proteins work are extremely important! According to an article on GEN, the discovery and understanding of anti-CRISPR proteins actually allows researchers to have greater control over gene-edits. In this article, Dr. Sontheimer, a professor in the RNA The RNA Therapeutics Institute at UMass Medical School, expressed how “CRISPR/Cas 9 is a good thing because it introduces specific chromosome breaks that can be exploited to create genome edits, but because chromosome breakage can be hazardous, it is possible to have too much of a good thing, or to have it go on for too long.” Anti-CRISPR proteins can be beneficial and work as an off switch for CRISPR, therefore advancing gene editing!




Crispr-Cas9: Coming to a Theater Near You

This sequel to GATTACA is to be released shortly, and this time, they’re transcending the movie screen and bringing the experience to reality!

Crispr-Cas9 is a fairly recent DNA-editing technique that has been developed, and allows for extremely easy and precise gene editing, a development said to be at least on par with PCR for bio engineering. In many ways, this is great. Now biologists won’t have to spend the time nor undergo the difficulty of creating variant DNA through old methods, meaning that all these cool genetic breakthroughs should be happening at an unprecedented pace! The problem is, it may be going too fast for humans to wrap their head around.

Similar to the ethical questions raised by the film GATTACA, countries and scientists are debating what regulations should be put on this new and powerful tool. With Crispr-Cas9, the possibility to genetically modify humans becomes a very real option to consider. Scientists could remove DNA sequences which lead to defects and diseases such as albinism and Huntington’s Disease. Or anything else, really.

(The miracle protein)

The main point of Crispr-Cas9 is not necessarily the ability it gives to scientists to easily modify DNA, but the increased rate at which we can understand what specific sequences of DNA do by altering them. Not only are we more able to modify DNA, we are now able to figure it out at breakneck speed.


Where it gets complex is, as always, how humans deal with it. Some people, such as Mark Leach, whose daughter has down-syndrome, believes that children with disabilities not only are still able to live rich lives, but also teach others to be more compassionate. Although debating if I would choose to let my child have down-syndrome or not for that reason seems like an absurd consideration, and most likely a coping mechanism, the point still stands that some people are uneasy with fixing genetic-related problems because “they wouldn’t be the same person.” (That’s the point!)

People are really afraid of change, aren’t they?


However, for those on the more lethal/completely disabling part of the genetic spectrum, the answer is more than clear.  Charles Sabine, the brother of the renown British lawyer John Sabine, who both have Huntington’s Disease at varying stages, says “If there was a room somewhere where someone said, ‘Look, you can go in there and have your DNA changed,’ I would be there breaking the door down.” Similarly, Matt Wilsey, a parent of a child with a terminal genetic illness, is awestruck at the ridiculousness of the situation: “As a parent with an incredibly sick child, what are we supposed to do — sit by on the sidelines while my child dies?” The oddity of the situation is, we have the capability to start figuring out how to solve these genetic issues with a very effective and efficient technique, it’s just that humans are riding the brakes, trying to slow down the almost inexorable progress of the freight train that is Crispr-Cas9. The irony is that many are afraid with tampering with the “sanctity” of human embryos. I would agree, except that humans defile it all the time. Birth defects, genetic diseases, miscarriages, etc. Of course, this is not intentional, but the parents have the largest hand in these outcomes, as they provide all the material,genetic and otherwise, to create the embryo, fetus, and eventually child. We are already making horrible mistakes with human embryo’s that cripple or kill the resulting child through the natural birth process. Personally, I would go off of this to say we should at least learn from this, so we could eventually progress far enough to prevent these things from ever happening, but I only ask all of the readers to keep this in mind: Nature (very badly) screws up too.


(The process Cas9 facilitates)

I’m not saying that we should be careless with this new and potentially dangerous or aberrant-spawning technology, but I think it’s time that humans come to terms with the fact that their world, and their lives, are entering a new era of existence. For millennia, structured humans have lived in a world where the outside world is the only thing we can manipulate, but now the very structure and formation of ourselves as well. I understand that such a change from a thousands-year-running viewpoint can be hard to make. We’ve never had to think about these things before as a species, because it wasn’t understood and out of our reach. It is daunting. It is terrifying. Only because it is unknown. But how are we to learn, to benefit, from this great potential, if we are too afraid to explore it? I understand that like any form of potential, it can go either way, but this is a great new time of possibilities that simply won’t go away, but reemerge constantly.

I think it’s time we gathered the courage to face it.

Biomedical Engineers paving the way for Immunology

For many years Biomedical Engineers have been attempting to find ways to make precise, efficient, and deliberate changes to the genetic material of living cells. Developments in this field can, not only help to eradicate many genetic diseases but it can also ensure what many scientists call “adaptive immunity”. With their newfound CRISPR – Cas9 technology, they may have found a solution to the problem that has been giving them so much grief


Adaptive Immunity occurs when a foreign body is recognized specifically for what it is and how it can harm the body. The other form of immune response is the innate response, in which there is a foreign body identified and the immune system sends any type of immune-response cell to general area to kill it. However, in adaptive immunity the body can individually recognize the problem and send exactly what needs to be sent, a much more efficient process.

Moreover, scientists hope that a cell’s ability to perform adaptive immunity will help contribute to eliminating harmful genetic mutations. Researchers hypothesize that, with this newfound technology, cells will be able to identify and respond to invading genetic material from a bacteriophage or invader of any sort. (quite possibly eradicating HIV and all other viruses from the Earth).

The science behind this new genetic-police force is as confusing as it is difficult to say… CRISPR…Cas9… what does any of that even mean?

CRISPR stands for Clustered Regulatory Interspaced Short Palindromic Repeats

Cas9 comes from the name of the protein-9 nuclease that scientists first found in Strep (Streptococcus Pyogenes) cells back in 2007 which help the bacteria participate in adaptive immunity.


All in all, its some pretty crazy and extremely complex stuff.

If you do so please, I suggest doing some of your own research on this topic if you have any questions. The opportunities afforded by this breakthrough are endless.


CRISPR Inhibited by Nucleosomes

CRISPR/Cas9 is currently being researched as a method to alter genes by editing or silencing them. This enzyme is derived from bacteria and archaea that use it to protect themselves from viruses. Researchers are currently finding more practical applications for this discovery. However, it has been recently been found that nucleosomes may play a large effect on CRISPR.

File:Nucleosome 1KX5 colour coded.png

Structure of a Nucleosome

At UC Berkeley, researchers have been studying the interaction of these prokaryotic enzymes with eukaryotic cells. They have found that nucleosomes may inhibit CRISPR/Cas9. Because bacteria likely do not use this enzyme to explore eukaryotic chromatin structures, their enzymes are not adapted to these types of structures. This is seen by many of the researchers’ experiments where stretches of DNA with low concentrations of nucleosomes had higher activity of CRISPR while others stretches with high concentrations of nucleosomes had lower activity. Scientists have also added chromatin remodeling enzymes while using CRISPR and found higher activity.

This has a few implications on the usage of the enzyme. While gene editing may be less influenced because only one cut is needed to introduce a sequence, scientists should take nucleosome concentration into account in gene silencing and epigenetic editing. CRISPR/Cas9 is an amazing discovery for genetics but we still have much to learn about how it works and how we can use it.

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