BioQuakes

AP Biology class blog for discussing current research in Biology

Tag: #CRISPR/Cas9 (Page 1 of 4)

Genetically Engineering the Food We Eat to Increase Consumer Desire

Solanaceae is an order of classification for a group of plants known as nightshades. The Solanaceae are a family of plants that ranges from annual and perennial herbs to vines, shrubs, and trees. Included in this family of variety are also a number of agricultural crops like tomatoes, medicinal plants like jimson weed, spices, weeds, and ornamentals. This group of plants are given the term “nightshade” because some of these plants prefer to grow in shady areas, and some flowers at night.Solanum americanum, fruits

The Solanaceae is one of humankind’s most utilized and important families. It contains some of the world’s most important vegetables as well as some of the most deadly toxic plants. Foods like potato, tomato, peppers, ground cherries, and eggplant all hail from this incredible plant. With the benefits of this plant family also comes the dangerous variety of plants. The belladonna, mandrake, Jimson weed, and tobacco also come from this family. Solanum trilobatum flowersNot only does this family of plants produce important vegetables and deadly plants, various chemicals and drugs can be harvested. Some of these include nicotine, solanine, capsaicin, atropine, scopolamine, and hyoscyamine.

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. It is a gene editing tool that can be used to edit DNA in cells. It used a specific enzyme called Cas9, which stands for CRISPR associated nuclease 9, and a specific RNA guide to either disrupt host genes or insert sequences of interest. CRISPR was initially used in bacteria as an adaptive immunity response but is now being used as an alternative in genome engineering.CRISPR illustration gif animation 1

In the agricultural world, plant breeding has always been the way to improve the traits wanted in a plant. With technological improvements, increased production has been vastly upgraded. Recent advances in gene editing have revolutionized the field of plant breeding. The process of genetic engineering has allowed people to target specific genes to improve rather than continuous breeding to produce the desired trait. 

Consumers choose the type of foods they want to eat by the traits of the fruit/vegetable, and in response, it leads the path to ensure that plant breeding will produce that trait again. In the horticulture industry, fruits are an important food that many people buy. Fruits are known to have a crucial source of energy, vitamins, fibers, and mineral components. The larger the fruit, the less sour and more nutrients it tends to store, influencing consumers to buy fruits that are bigger in size and shape. As a plant family with various crops, Solanaceae crops have a variety of fruit sizes and shape features. With advancing gene editing technology, Solanaceae fruit crops have been on the receiving end of being genetically modified to increase desirable traits of fruit size, fruit weight, fruit quality, and plant architecture.Maduración del tomate (Solanum lycopersicum)

Many of the vegetables and fruits we eat today are slowly being improved with CRISPR. For instance, in tomatoes, the ARGONAUTE7 (SlAGO7) gene function in leaf shape development was one of the first edits done with CRISPR Cas9. Tomatoes have been at the forefront of CRISPR Cas9 gene editing on plants because it is a model crop that is able to grow variability. Many more plants of the Solanaceae family, like the goji berry and groundcherry, have been engineered to produce the best product and CRISPR gene editing will continue to enhance the fruit and plant.

This CRISPR gene editing research on the order of Solanaceae plants is relevant to AP Biology because of gene editing. In the first year of biology, we learned about the taxonomy of species and the order of specificity. The order of Solanaceae plants indicates that it isn’t a particular family of plants that includes the different genus and species. Instead, it is a broader classification. We didn’t specifically learn about CRISPR gene editing in class this year, but we learned about DNA and RNA and their replication process. In a way, we learned about CRISPR because it relies on a strand of RNA with the preferred traits that is then transcribed into DNA.

Cas9/10

Gene editing sounds to most like an intriguing opportunity at the very least, if not a groundbreaking advancement in human development, however it does not come without any flaw. We are not living in “Gattaca”quite yet to say the least. One of the most common gene editing processes, CRISPR, is equipped with a relatively predictable flaw in particular; an error taking place at the molecular level that results in the wrong genome being altered than what had been intended, therefore leading to potentially dangerous or life altering mutations in said gene. A team of specialized professionals at the University of Texas at Austin decided to revamp a significant component used for the CRISPR gene editing process. Their new version of Cas9 reduces the chances of the wrong genome being manipulated by thousands.  This is a figurative unicorn of scientific discovery,  it is groundbreaking on top of groundbreaking, it is cloth cut from the fabric of similar discoveries that have changed the course of human history and still – it is only the beginning.

When there is an error in the way the genomes are adjusted, it is a rather simple explanation as to how, and even simpler when describing how the new version of Cas9 can fix it. When the letters making up the DNA’s structure are incorrectly assembled or mismatched, causing a lack of stability in the structure of the DNA itself. Due to this, Cas9 is not capable of making the necessary adjustments to the DNA in order to properly execute the procedure. The new version of Cas9 is far more capable and strong, meaning that it can in fact execute the procedure.

Although this new Cas9 is an answer to a previously inherent setback to gene editing, it doesn’t come without its own respective setbacks. A primary caveat to the increased accuracy of this Cas9 is that it works at a much slower pace than Cas9 that is naturally occurring.

There is a self awareness that seeps through this accomplishment to the people that set it in motion. Kenneth Johnson, a professor at the University of Texas at Austin and co-author of the study even says that this newfound tool “could really be a game changer” when it comes to further use of gene editing among the public. It is truly a tremendous feat conquered by this group of experts in the field of genetic engineering.

Ultimately, further advancements in genetic editing could very well change the human race and the world as we know it so long as quality time and effort is put into it, as seen with this study. With the incentive of the potential advancement of human kind as a whole, its anyone’s guess as to what could one day be possible.

Can we make Jurassic Park real?

CRISPR technology has already demonstrated its potential to revolutionize modern biology. Summarized, CRISPR is a gene editing technology. It has the ability to change the sequence of DNA in living cells, therefore changing their traits. However, the applications of CRISPR extend far beyond simple fun with gene editing. CRISPR can be used to modify the foods we eat, making them easier to grow and more resistant to harsh climate. CRISPR has even been theorized to have implications for treating human genetic diseases. However, how far does this technology go?

Dino Park

A group of scientests have been focusing on a much more radical side of CRISPR: they are attempting the revival of an extinct species. The Christmas Island Rat went extinct over 100 years ago in 1903. Thankfully, some DNA of the rat has been maintained, allowing scientists to sequence the genome. Through analysis, they have found that the Christmas island rat is very closely related to the brown rat. In fact, the genomes have a 95% similarity between them. This similarity begs the question, can we CRISPR a Brown Rat into a Christmas Island Rat?

Because of the highly similar genomes, scientists believe that they can use the gene editing technology in CRISPR to recreate the Christmas Island Rats from the brown rat. While they have not yet achieved their goals, they are confident in their ability to produce results. Although modifying a rat to bring back a close relative is a long way off from bringing back dinosaurs from nothing, this amazing experiment may pave the way for future scientists to make the movies real life. As science progresses, we may be able to transform more complex and distantly related species, we will just need to wait and see.

CRISPR Quits Coronavirus Replication

The gene-editing CRISPR has now been utilized by scientists to prevent the replication of Coronavirus in human cells, which can ultimately become a new treatment for the contagious virus. However, since these studies were performed on lab dishes, this treatment can be years away from now.

Firstly, CRISPR is a genome-editing tool that is faster, cheaper, and more accurate than past DNA editing techniques whilst having a much broader range of use. The system works by using two molecules: Cas9 and guide RNA (gRNA). Cas9 is an enzyme that acts as “molecular scissors” that cuts two strands of DNA at a specific location. gRNA binds to DNA and guides Cas9 to the right location of the genome and ultimately makes sure that Cas9 cuts at the right point.

 

CRISPR'S Cas9 enzyme in action

CRISPR’S Cas9 enzyme in action

In this instance, CRISPR is used to allow the microbes to target and destroy the genetic material of viruses. However, they target and destroy the RNA rather than the DNA. The specific enzyme they use for this is Cas13b, which cleaves the single strands of RNA, similar to those that are seen in SARS-CoV-2. Once the enzyme Cas13b binds to the RNA, it destroys the part of the RNA that the virus needs to replicate. This method has been found to even work on new mutations of the SARS-CoV-2 genome, including the alpha variant.

COVID-19 vaccines are being distributed around the world, but an effective and immediate treatment for the virus is necessary. There are many fears that the virus will be able to escape the vaccines and become a bigger threat. Although this treatment is a step in the right direction for effective treatment of COVID-19, this technique will ultimately take a long time for the treatment to be publicly available.

CRISPR is greatly relevant to AP Bio, as seen through its use of enzymes in DNA replication. CRISPR utilizes Cas9, an enzyme that is similar to helicase. In DNA replication, the helicase untwists the DNA at the replication fork, which after the DNA strands are replicated in both directions. 

This article is fairly outdated since there have now been immediate treatments created for COVID-19. But, what do you think about the use of CRISPR for future viruses and pandemics? Personally, I believe that CRISPR will ultimately become a historical achievement in science due to its various uses. Thank you for reading and let me know what you think in the comments! 

 

A Vision For a Better Future

CRISPR is a world changing technology that is essentially used to edit genes. The discovery of CRISPR took place in the University of Alicante, Spain. Reported in 1993, Francisco Mojica was the first to characterize CRISPR locus. Throughout the 90s and early 2000s, Mojica realized that what was once reported as unique sets of repeat sequences actually shared common features, which are known to be hallmarks of CRISPR sequences. Through this finding, Mojica was able to correctly hypothesize that CRISPR is an adaptive immune system. In the year 2013, Feng Zhang, was the first scientist to successfully adapt CRISPR-Cas9 for genome editing in Eukaryotic Cells. Zhang was able to engineer two different Cas9 orthologs and he then demonstrated targeted genome cleavage in both human and mouse cells. They discovered that this system could then be used to target multiple genomic loci and could also drive homology directed repair.

CRISPR-Cas9 mode of action.png

How Does it Work?

“Clustered regularly interspaced short palindromic repeats,” also known as CRISPR, are repeats found in bacteria’s DNA. CRISPR-Cas9 was adapted by scientists from a naturally occurring genome editing system in bacteria. This bacteria captures parts of DNA from invading viruses and it uses them to create DNA segments known as CRISPR arrays. This DNA allows the bacteria to recognize and remember the virus’s. If the same virus, or a similar one, attacks again, the bacteria will consequently RNA segments in order to target the viruses DNA. After, the bacteria uses the enzyme Cas9 in order to cut the DNA apart, thus disabling the virus. Scientists in a lab will create small pieces of RNA that attach to a specific target sequence of DNA and also the Cas9 enzyme. In this process, the RNA is used to recognize DNA and the Cas9 will cut the targeted DNA. Once cut, researchers will utilize the cell’s ability to repair DNA in order to add or remove pieces of genetic material. It can also replace existing DNA with custom DNA in order to make changes.

How is it used?

CRISPR is a tool that can be used to fight cancer among other known diseases. The therapy involves making four modifications to T-cells. T-cells are cells that help fight cancer. CRISPR adds a synthetic gene that gives the T-cells a claw-like receptor. This receptor can locate NY-ESO-1 molecules on cancer cells. CRISPR is then used to remove three genes. Two of the removed genes can interfere with the NY-ESO-1 receptor and the third limits a cell’s cancer killing abilities.

Another way CRISPR is used is against Leber’s Congenital Amaurosis(LCA). LCA is a family of congenital retinal dystrophies that results in vision loss. Patients tend to show nystagmus, sluggish pupillary responses, decreased visual acuity and photophobia. The CRISPR trial focuses on one gene mutation that causes a severe form of degeneration. It is said that this mutation creates somewhat of a “stop sign,” and RNAs will target sequences on either part of the stop sign. The Cas9 enzyme will then cut them out, allowing the DNA to then repair itself.

Instead of Bringing Back Dinosaurs, These Scientists are Bringing Back the Extinct Christmas Island Rat

Majestic dinosaurs and mammoths on our planet both underwent extinction millions and millions of years ago. The Christmas Island rat? In 1908. De-extinction techniques, also known as resurrection biology, garnered popularity within the science world in the 1990s. The Encyclopedia Britannica defines it as, “the process of resurrecting species that have died out or gone extinct.” Here is how these scientists are attempting to bring back a rat species that you have probably never heard of, and what that can mean for the future.

De-extinction using CRISPR gene-editing

 

File:MaclearsRat-PLoSOne.png - Wikimedia Commons

path of extinction of the Christmas Island rat

The process of de-extinction with the Christmas Island rat is driven by the method of CRISPR gene-editing, which allows for the genome of organisms to be modified, or edited, meaning that an organism’s DNA can be changed by us humans. This allows for genetic material to be added, removed, or modified at specific locations said genome. The idea behind the de-extinction of an animal through CRISPR gene-editing is to take surviving DNA of an extinct species and compare it to the genome of a closely-related modern species, then use CRISPR to edit the modern species’ genome in the places where it differs from the extinct one. The edited cells can then be used to create an embryo implanted in a surrogate host.

CRISPR thought to be “genetic scissors”

Thomas Gilbert, one of the scientists on the team of this project, says old DNA is like a “book that has gone through a shredder”, while the genome of a modern species is like an intact “reference book” that can be used to piece together the fragments of its degraded counterpart.

What is the difference between a genome and a gene?

File:Human genome to genes.png

Gene depicted within genome

Genes, a word you are most likely familiar with, carry the information which determines our traits, or features/characteristics that are passed on to us from our parents. Like chromosomes, genes come in pairs. Each of your parents has two alleles of each of their genes, and each parent passes along just one to make up the genes you have. Genes that are passed on to you determine many of your traits, such as your hair color and skin color. Known dominant traits are dark hair and brown eyes, while known recessive traits are blonde hair and blue or green eyes. If the two alleles that you receive from your parents are the same, you are homozygous for that gene. If the alleles are different, you are heterozygous, but you only express the dominant gene.

Each cell in the human body contains about 25,000 to 35,000 genes, and genes exist in animals and plants as well. Each gene is a small section of DNA within our genomes. That is the link between them, and they are not the same.

Is this possible? Can we really bring back the dead?

Reconstructed image of the extinct woolly mammoth

See, CRISPR gene-editing itself is of great interest for having shown promising results in terms of human disease prevention and treatment for diseases and single-gene disorders such as cystic fibrosishemophilia, and sickle cell disease, and shows promise for more complicated illnesses such as cancer, HIV infection, and mental illness–not so much with de-extinction. Here’s a simple diagram displaying the process.

File:Crispr.png

In this scenario, it is not looking very likely that these rats can come back. Gilbert and his team of 11 other scientists, through extensive processes and attention to small-detail, have in total reconstructed 95% of the Christmas Island rat genome. While 95% may be an A on a test, in regards to genomes, that 5% is crucial. In this case, the missing 5% is linked to the control of smell and immunity, meaning that if we were to bring this animal back, it would lose key functionality. Gilbert says 100% accuracy in genome reconstructing of this species is “never” going to happen.

The success of de-extinction is quite controversial in itself. Restoring extinct species can mean an increase in biodiversity and helping out our ecosystems which are suffering greatly from climate change.  However, research also suggests it can result in biodiversity loss through possibly creating invasive species (yes, I wrote this) or for other reasons.

While the science is interesting, the reality of the unlikeliness of de-extinction becoming a normal and official process is kind of dream-crushing. Who knows, maybe as technology advances, hopefully, we can make all of this happen without harmful side effects, aid our ailing ecosystems, and visit some mammoths on a safari vacation!

Away With Treadmills and Low Carb Diets: Is CRISPR the New Hack For Fat Loss?

Are you sick and tired of spending all of your time running on the treadmill and eating restrictive diets? Are you looking for a way to hack fat loss without ruining your way-of-life? Look no further than CRISPR gene-editing!

In humans, stubborn body fat can be attributed to either white or brown fat. Brown fat, specifically, is used in humans primarily for insulation, and can be tapped into when we are cold or need to ramp up our metabolism to generate heat. This fat is caused by a caloric surplus in humans, and is burned off by engaging in caloric deficit. However, in mice studies conducted by Steven Romanelli, Ormand MacDougald, and colleagues, CRISPR gene-editing offers promising results regarding the topic of brown fat loss in humans. CRISPR-Cas9 Editing of the Genome (26453307604)

But what exactly is CRISPR gene-editing? CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeat, gene-editing entails organizing short, palindromic DNA sequences of bacteria. These DNA sequences are surprisingly important in the immune function of these bacteria and other microorganisms, making CRISPR an incredibly promising and innovative tool in science research. 

Bacteria have short sequences of variable DNA called “spacers” in between CRISPR DNA sequences. This DNA helps protect the bacterium from reinfection from viruses. If any virus were to attack the bacterium, the CRISPR DNA sequences would cut up that viral DNA matching any spacer within the genetic code of the bacterium, preventing it from reinfection. 

CRISPR gene-editing works by processing invading viral DNA into short fragments that are inserted into the CRISPR DNA as spacers. Then, CRISPR replicates and spacers in the DNA of the bacterium experience transcription, in which DNA becomes RNA and CRISPR RNAs. These CRISPR RNAs help bacteria kill viruses, as they match the exact DNA as the viral DNA attacking the bacterium. 

In mice experiments conducted by Romanelli, MacDougald, and colleagues, has used CRISPR gene-editing to have an enzyme named Cas9 break strands of DNA and a single piece of RNA to be packed into a harmless virus cell that will be delivered into cells in the study, which are brown fat cells in this case. This process has shown to delete several genes, namely the UCP1 gene in mice, that allows brown fat to exist and create heat. However, the mice in the study did not die when exposed to cold environments. They were able to survive despite a huge loss in brown fat. 

Accordingly, using CRISPR gene-editing as a tool for brown fat loss in humans provides incredibly promising results. It is certain that, once CRISPR gene-editing becomes available for use in the reduction of brown fat in humans, I will no longer be using the treadmill as my mode of fat-burning and shift toward this method instead.

Potato, Patato No More? Scientists have cracked the code to diversifying the classical starch

With gene-editing technologies such as CRISPR, the variety in produce has been growing at greater rates than ever. It seems as if it is only a matter of time until we get a talking pepper. However, potatoes have been lagging behind. A potato may look quite simple to the human eye, but it is actually quite complex in the world of genomes. For this reason, the human genome was discovered more than 20 years before the genomes that make up a delicious fast food French fry.French Fries

So what is it that makes the potato genome puzzle so difficult to crack? Human offspring receive one of each chromosome from the mother and father, while potatoes receive two of each chromosome from each parent. This results in 4 total copies of each chromosome and in turn four copies of a given gene. A species such as this is called tetraploid. The increase in genes per trait makes editing a given trait that much more difficult. Another task of great difficulty is recreating the potato genome. A task much more difficult than doing so for humans. 

 

Haploid, diploid ,triploid and tetraploid

Scientists Korbinian Schneeberger and Hequan Sun found a clever shortcut. They realized that the pollen cells of potatoes, similar to gametes in humans, contain only half the chromosomes of a body cell. Pollen cells are by this logic, diploid cells containing two of each chromosome. Sequencing the DNA of large amounts of pollen cells allowed the scientists to map out the full genome of a potato. The construction of this genome will make identifying and editing diverse variants of potatoes a much easier task. 

This begs the question of why? Why do we need variety in species of potato? Historical events such as the Irish potato famine of  1840 are a prime example of the importance of produce variety. The famine was caused by tuber blight. A potato is a tuber, a storage stem of plats, and blight is a plant disease commonly caused by fungi. Despite being the most important crop and source of food at the time in most of Europe, the incredible lack of variability of a potato meant no species of potato was resistant to the disease. With concerns over climate change, and an increase in potato popularity; “The potato is becoming more and more integral to diets worldwide including even Asian countries like China where rice is the traditional staple food. Building on this work, we can now implement genome-assisted breeding of new potato varieties that will be more productive and also resistant to climate change — this could have a huge impact on delivering food security in the decades to come.” (Max Planck Institute for Plant Breeding Research), make this issue more important than it may appear. 

The Redesigning of CRISPR

Cas9, a key component of a widely used CRISPR-based gene-editing tool has been redesigned by scientists at The University of Texas at Austin to be thousands of times less likely to target the wrong stretch of DNA while remaining just as efficient as the original version. This could potentially make gene replication safer and more abundant for medical use.

The CRISPR-Cas9 system consists of an enzyme that introduces a change or mutation into DNA. Cas9 enzymes can cut strands of DNA at a specific location in the genome so parts of the DNA can then be added or removed. CRISPR-based gene-editing tools are adapted from naturally occurring systems in bacteria. In nature, Cas9 proteins search for DNA with a very specific sequence of 20 letters. When most of the letters are correct, Cas9 could still change these DNA fragments. This is called a mismatch, and it can have disastrous consequences in gene editing.

The challenge with using CRISPR-based gene editing on humans is that the molecular machinery occasionally makes changes to the wrong section of a host’s genome. This could possibly repair a genetic mutation in one spot in the genome but may accidentallyDna, Analysis, Research, Genetic Material, Helix create a dangerous new mutation in another.

SuperFi-Cas9 is the name of the new version of Cas9 which has been studied and proven to be 4,000 times less likely to unnecessarily cut off-DNA sites but operates just as fast as naturally occurring Cas9. In the Sauer Structural Biology Lab, scientists were surprised to discover that when Cas9 encounters a type of mismatch, there is a “finger-like structure” that swoops in and holds on to the DNA, making it act like the correct sequence. Usually, a mismatch leaves the DNA unorganized since this “finger-like structure” is mainly used to stabilize the DNA. Based on this insight, scientists redesigned the extra “finger” on Cas9 so that instead of stabilizing the part of the DNA containing the mismatch, the finger is stored away which prevents Cas9 from continuing the process of cutting and editing the DNA. This result in SuperFi-Cas9, a protein that cuts the right target just as readily as naturally occurring Cas9, but is much less likely to cut the wrong target.

This applies to our unit on mitosis when cells are replicating DNA in the S-phase. When chromosomes are duplicated, gene replication occurs. Sometimes gene replication could result in mutations which could lead to a cell not functioning properly. A cancerous cell is an example of cell not performing normally since it rapidly performs mitosis causing the cell to duplicate uncontrollably. This results from an abnormality in gene replication where CRISPR technology can locate this mutation and restore the cell back to normalcy.

Cell Cycle Regulation in Revolutionary Gene Editing Technique (a.k.a. CRISPR)

There are more than 500 different types of human cancers. Wouldn’t it be wonderful if scientists could develop cures for all of them? Scientists believe that CRISPR gene-editing can be used to cure some cancers. CRISPR (an acronym for clustered regularly interspaced short palindromic repeats) is a way of targeting a specific bit of DNA inside a cell which can then be gene-edited to change such bit of DNA. CRISPR has also been used for other purposes, such as turning genes on or off without changing their DNA sequence.

 

Recent research has found a link between CRISPR gene-editing and mutated cancer cells. Scientists believe that a further understanding of this link can identify a group of genes which should be monitored for mutations when cells are subjected to the CRISPR gene-editing method. Although CRISPR gene-editing holds promise for cell repair, the application of CRISPR gene-editing, which is meant to identify and correct damage in cells, can also cause damage to cells in a controlled manner. Such damage activates a protein, p53 (“also known as the guardian of the genome”), which helps repair damaged DNA. 

CRISPR-Cas9 mode of action

P53 is a transcription factor, which is a protein that regulates the rate at which DNA is transcribed into RNA. These transcription factors bind to regulatory sequences in proteins, thus changing the shape of DNA, ultimately making them the most vital form of gene regulation. Transcription factors include many proteins but exclude RNA polymerase, which pries two strands of DNA apart and joins two strands of DNA together (Campbell, 280). P53 works by sliding along the damaged DNA, seeking a critical site to which it attaches and then sends a message to halt cell division until the DNA is repaired. In other words, p53 acts as a checkpoint in the cell cycle, preventing cell from proceeding though the G1 and G2 phases of the cell division cycle. In mice, the same exact transcription factor exists; those that lacked the Trp53 gene developed tumors at a far faster rate than those with the functioning gene.

 

By using CRISPR technology to damage DNA at the same cite at which DNA damage occurs, scientists are able to identify the protein responsible for cellular proliferation. If damage to the cell is too severe then p53 triggers apoptosis (the death of cells which occurs as a normal and controlled part of an organism’s growth or development) so that the damaged cell is destroyed. However, sometimes p53 is itself damaged which prevents such protein from binding to the damaged DNA in order to repair it or otherwise signaling destruction of the cell. When this occurs, the damaged cells multiply and grow, resulting in tumors. Scientists have found alterations in p53 in more than half of all cancers and thus, consider p53 the most common event in developing cancer.

 

New studies show that p53 inhibition can make CRISPR more effective thus, counteracting “enrichment” (the process of purifying cells for downstream applications such as qRT-PCR, cell polarizations ex vivo, or to enrich cells for use in a flow cytometry experiment) of cells with p53 mutations which has been observed to occur in cell cultures when such cells have been subjected to CRISPR. In other words, there is in vitro evidence that CRISPR technology causes harmful p53 mutations to be more prevalent in the population that has been subjected to the CRISPR technique. These findings suggest that there is a group of genes that should be monitored for mutations when the CRISPR gene-editing method is applied to cells. 

 

Cancer is a devastating disease that has taken the lives of many people. Members of my family have suffered and lost their battle to cancer (most recently my dear aunt this past weekend). CRISPR presents the possibility of finding cures to cancer which are specifically designed to target the particular genetic mutations that are unique to each individual. Perhaps, the cure to cancer will be achieved sooner than we realize,  although clearly not soon enough. 

 

Works Cited:

Reece, Jane B, and Neil A. Campbell. Campbell Biology. Boston: Benjamin Cummings / Pearson, 2011. Print.

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?

CRISPR Causes Cancer, Sort Of

          Scientific researchers are always looking for ways to improve modern science and help create new treatments. Currently, CRISPR, “a powerful tool for editing genomes,” holds the ability to help advance medicine, specifically gene editing, so long as the kinks in this specific method are worked out. One of these problems is the DNA damage caused by CRISPR “activates the protein p53,” which tries to protect the damaged DNA. This raises not one, but two concerns as present p53 can diminish the effectiveness of this technique, however when there is no p53 at all cells grow rapidly and become cancerous. As we learned in AP Biology class, typical cells communicate through chemical signals sent by cyclins that ensure the cell is dividing the right amount. Cancer cells, however, contain genetic mutations that prevent them from being able to receive these signals and stop growing when they should. “Researchers at Karolinska Institute” have discovered that “cells with inactivating mutations of the p53 gene” have a higher survival rate when contingent on CRISPR. To further their research, they discovered genes with mutations similar to those of the p53, and also “transient inhibition” of the gene could help prevent “the enrichment of cells” that are similar. Although seeming antithetical, these researchers proved that inhibiting p53 actually makes CRISPR work better and prevent enrichment of mutated p53 and other similar genes. 

CRISPR logoDNA animation

           These results give crucial information, helping advance CRISPR and make it more usable in current medicine. Additionally, the researchers have uncovered the possibility that the damage CRISPR causes to DNA might be key in creating a better RNA sequence (the RNA sequence tells us the “total cellular content of RNAs”) guide, showing where DNA should be changed. In future tests, these researchers want to try and get a better idea of when the enhancement of mutated p53 cells from CRISPR becomes a problem.    

CRISPR: A Possible Solution to Genetic Diseases?

A few decades ago in science fiction, there were talks of things like genetic modification in babies. This was more along the lines of creating the ‘perfect’ human, rather than using genetic modification to stop certain genetic illnesses. An example of this is in the 1997 movie, Gattaca, where we see an unmodified (genetically) person struggle to live in a world of genetically modified people. While it is fiction, it showed how being able to alter someone’s genetic flaws can go a long way. Despite, at the time, this seeming to just be science fiction, some of these concepts of gene alteration might become reality. These concepts becoming reality would all be due  to CRISPR.

CRISPR logo

Some of you might be thinking, “what is CRISPR?,” and that’s okay because before researching it I was thinking the same thing. CRISPR is a type of genetic engineering technique in molecular biology. This technique allows for the modification of genomes in a living organism. This technique is actually based off of CRISPR-Cas9 antiviral defense system, which can cut genomes. This has inspired CRISPR to contain Cas9 nuclease complexed with gRNA. This is the sent into a cell and is able to cut a cell’s genome at a certain position. This allows for specific genomes to be removed, as well as allowing new ones to be added. So in summary, CRISPR is a method of removing certain genomes of a cell, and in some cases replacing and/or adding a genome as well.

Now that CRISPR has been explained and we know what it is as well as how it works, we are now able to look at studies involving it. While CRISPR seems great and all, Heidi Ledford posted an article about how the use of CRISPR in embryos can cause some unwanted changes to the embryo. While experimenting, researches found that the use of CRISPR on an embryo can not only cause unwanted changes at the genome target site, but it can also cause changes near the genome target site. While some of you may think that the pros out-weigh the cons in this instance, geneticist Gaétan Burgio states that, “the on-target effects are more important and would be much more difficult to eliminate.” The on-target effects (negative) are so bad that it may not be worth doing even if it is to eliminate genetic diseases.  The idea that the cons outweigh stopping a genetic disease shocked me, as in our biology class we talked about genes and genetic diseases, and how even though they can be extremely rare, they can be irreversible, life changing, and in some cases fatal. This rejects the idea that the pros could out-weigh the cons, which puts a pin in this genetic modification breakthrough.

After looking at CRISPR as well as the research shown on genetic modification of embryos, I have realized how far we still are from elimination of genetic diseases. Despite issues arising in the experiment, I hope that they can put CRISPR to good work in order to stop the seemingly impermeable genetic diseases. And who knows, if we can master genetic modification with CRISPR, the ideas presented in Gattaca could soon seem like reality.

 

 

Could This New Gene Editing Technique Increase Burger Supply?

A gene editing technique by the name of CRISPR is a very important and useful tool in the scientific world of genetics. CRISPR is essentially a way for scientists to edit genes which is becoming useful in many different studies such as cancer research. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. The process contains two main components: a Cas9 protein and a guide RNA. The Cas9 protein’s job is to cut DNA while the guide RNA is what recognizes what needs to be edited. Once a specific guide RNA is created for a specific part of the DNA that scientists want to edit, it is then attached to Cas9. This complex is then added to theVaca (Bos primigenius taurus), Tierpark Hellabrunn, Múnich, Alemania, 2012-06-17, DD 02 target cells where it cuts finds and cuts the matching DNA portion to then be edited. This gene editing process is not only used on human cells, but animal cells as well.

U.S. regulators have now said that certain cattle produced through CRISPR are going to be able to be raised for the production of meat. These specific cattle are called the PRLR-Slick cattle which are among a few of the CRISPR animals that are approved for food purposes. These specific cattle were the first to receive a “low-risk determination for enforcement discretion”. When looking at the specific gene editing that was done to the cattle it was very similar to the natural mutations that occur when cattle are placed in hot climates. They were therefore able to conclude that these cattle who were short-haired cattle were the same as the non edited cattle with the same hair mutation trait (caused by CRISPR). The company who produced the cattle, Acceligen, claim they produced the cattle in order to tolerate hot weather better.

CRISPR Causes Cancer? Or Does It?

CRISPR illustration gif animation 1

The ground-breaking scientific break through of gene editing is finally here with the technique called CRISPR. CRISPR, or gene cutting, is the method of cutting a strand of DNA and letting the DNA repairer function repair the cut by itself. But it is at this instant where scientists introduce some changes to the genes, which the DNA will reproduce; DNA naturally grows back the mutated Genes. This CRISPR sounds great in the world of science, where certain genes can be modified onto a person. Though there are different opinions on gene-modifying, CRISPR has yet to be fully perfected. One of these hurdles is P53; a tumor suppressing protein. P53 is known as the “guardian of the genome” as this protein determines “whether the DNA will be repaired or the damaged cell will self-destruct” (MEDICINEPLUS). While this is good news, it is observed that “a dataset of >800 human cancer cell lines identified additional factors influencing the enrichment of p53-mutated cells.”(aacrjournals). So a lot of cancer cells have had a mutated P53 protein. So why does this happen? Why is the guardian of the genome being mutated?

 

P53 mutations are “missense mutations,” meaning they mutate in relation to a gene being edited, which creates different amino acids. By creating a whole new set of amino acids, the cell completely changes and in this case, mutates the P53. By mutating the P53 protein, P53 can no longer stop the cell division cycle, immensely increasing the chance of cancer cells. Although I make CRISPR sound like a dangerous operation to do(*I am not a scientist*), Researchers at Karolinska Institutet say to” have found new links between CRISPR, p53 and other cancer genes that could prevent the accumulation of mutated cells without compromising the gene scissors’ effectiveness.”

Another new research point mentions that although a lot of P53 mutations occur when subject to CRISPR, “cells with mutations are there from the start.” This is still a huge unknown to scientists, as CRISPR does cause P53 to mutate, there are already mutated P53 cells beforehand. Though this does prove that P53 is affected by other factors instead of only affected by CRISPR. Scientists still have much to uncover about gene-editing and in the future, they could possibly change somebody’s genes for a good cause.

 

 

 

Researchers at UT Austin tweak cas9 to make CRISPR gene-editing 4,000x less error-prone

A huge stride in ensuring the efficacy of CRISPR genome-editing has been made by researchers at the University of Texas at Austin. The CRISPR gene editing tool is a new genetic engineering technique that can, by using an enzyme called Cas9, correct problematic genomes in a person’s DNA. It finds the genome that its programmed to and cuts it out of the DNA, leaving the organism without that DNA, and inhibiting the organism from spreading that gene to their offspring. There have been studies have shown CRISPR has been effective in editing genomes that may cause disease. In a study where the Cas9 enzyme was injected into the bloodstream of six people with a rare and fatal condition called transthyretin amyloidosis, those who received the higher dose saw a decline around 87% in production of the misshapen protein that causes this condition.

For many diseases, Gene therapy is the “Holy Grail”. For treatment of Sickle-Cell Anemia, CRISPR has been thought of as a definitive cure. In 2017, it was reported that a 13-year-old boy with HbSS disease had been cured with gene therapy. This treatment also allows the carrier of this gene to reproduce without any risk of their offspring being affected by SCD.

GRNA-Cas9However, there are concerns that when performing the genome editing, the wrong segment of DNA could be targeted by scientists and removed, resulting in potentially drastic consequences. Another concern is that editing out certain genes is societally damaging, as it is considered unnatural to be able to edit the genomes of human bei

ngs. Another major safety concern is mosaicism (when some cells carry the edit but others do not); this could result in many different side effects. Due to the many uncertain aspects around the danger of genome-editing, there has been delay in passing legislation approving genome-editing.

 

In a study published on March 2nd 2022 at the University of Texas at Austin, researchers have found a previously unknown structure in the Cas9 protein that is thought to attribute to these genetic mistakes. When using cryo-electron microscopy to observe the Cas9 protein at work, the researching team noticed a strange finger-like structure that stabilized the off-target gene section to be edited instead of editing the target gene.

The researchers at the University of Texas at Austin were able to tweak the protein, preventing Cas9 from editing the wrong sequence. This change has made the tool 4,000 times less likely to produce unintentional mutations; the team calls the new protein ‘SuperFi-Cas9’.

While other researchers have made similar edits to make the Cas9 protein more accurate in its editing, these often result in slowing down the genome editing process. At UT Austin, the researchers say that SuperFi-Cas9 still is able to make edits at the normal speed.

The researchers plan to test SuperFi-Cas9 further in living cells as opposed to the testing thats been done with DNA in test tubes. Hopefully they’re able to cement the accuracy of SuperFi-Cas9, and that this may accelerate us on our way to implementing CRISPR gene editing in the current medical world. Let us know in the comments below what your thoughts are on CRISPR editing, and if you think we should continue researching it!

Can Gene Edited Tomatoes Save Your Life?

In a new invention by Hiroshi Ezura, the chief technology officer at Sanatech and molecular biologist, Tomatoes can now lead to lower blood pressure and higher relaxation.

This invention is based off a new phenomenon that is becoming more and more popular in Japan, food that is genetically edited using CRISPR technology. This technology is used to increase the amount of GABA in foods. GAMA, or gamma-aminobutyric acid, is a neurotransmitter and amino acid that “blocks impulses between nerve cells in the brain” (Waltz, Scientific American).

GAMA is being tested by many groups, including Ezura and his crew, for positive correlations between available GAMA, and health benefits. So far, there have not been any confirmed guaranteed health benefits, but the data from other genetically modified foods shows generally there are health benefits. People eating the food should feel more relaxed. Other tests have been done in the human, or animal bodies. GAMA is a natural substance found in humans, so the genetic mutations do not add a foreign substance to the body – it is safe to consume.

CRISPR technology is at the heart of this. This is a genetics technology in which one can add, take away, or alter sections of DNA. DNA is a double helix which consists of the nucleotide bases, Adenine, Cytosine, Guanine, and Thymine. When certain sections of the genetic code, represented in letters, are replaced by others, certain genes can change. Genes are certain pieces of DNA that carry genetic information which can alter how someone looks or functions. When worked on a tomato, it is able to alter a gene that gets rid of a pathway called the GABA shunt, which through a series of events limits GABA in cells.

Basepairs Graphic Public Domain

This technique has been used before, but it is so special because this is the first time it has been a commercial food product. This is exciting; genetic engineering can have negative effects in some areas, but so far the data shows that it is effective. Personally, I hope there is a rigorous series of tests that has to be conducted in the future for each CRISPR modified food to be commercially produced and sold.

A New Mosquitoes Exterminator: CRISPR

Mosquitoes might be just pesky little insects that might start appearing again in a few months. They leave their saliva in your skin, and this causes an itchy bump. But in other parts of the world. Estimates report that mosquitoes have killed up to 52 billion people in history by spreading malaria, yellow fever, and dengue. It is currently killing 1 million people per year, even with advanced medicine and healthcare around the world. Doesn’t that horrifying statistic shock you? 

Aedes aegypti

A close shot of Aedes aegypti

To drastically lower this number, researchers took a smart approach and decided to eliminate the source of the problem. They decided to use CRISPR to genetically alter the male flies so that they become sterile.  Professor Craig Montell from UC Santa Barbara altered the gene of Aedes aegypti, the main type of mosquito that transmits dengue, yellow fever, Zika, etc. 

Previously, scientists just radiated and applied chemicals to sterilize male mosquitoes in hopes to alter their genes since “there are enough genes that affect fertility that one will likely be altered,” making them infertile. However, this would leave, many of the mosquitoes to be sick and die prematurely since other genes that don’t relate to fertility are also changed.

Using CRISPER/Cas9, researchers removed B2t, a gene that specifically affects male fertility in mosquitoes. Unlike in previous efforts, the sterile mosquitoes were completely healthy. 

This whole effort to sterilize insects is part of a greater method called the sterile insect technique (SIT). Scientists release way more sterile insects than there exist in the wild. The population will crash as females will not be mating with a lot of males that are capable of making offspring. A benefit of releasing males instead of females is that males feed on nectar, not blood,  so it will not cause major disturbance to communities.

To sexually reproduce, a sperm cell must meet an egg. Each gamete is a haploid that has a single set of chromosomes. The sperm and egg combine to produce a zygote making it a diploid with a complete set of chromosomes. If a male is sterile, then they are not able to produce or release their sperm, making it impossible for those insects to reproduce.

The effect of this technique is more effective after each cycle, so when you release the same amount of mosquitoes after 3 cycles, the population change will be way more drastic. A downside to this is that sterile male mosquitoes need to be reintroduced after they die off since they cannot pass on their mutated gene.

Although researchers have successfully identified a way to isolate the gene and remove it to make male mosquitoes fertile, they still needed to find the optimal ratio of lab mosquitoes to wild type to ensure that they do not wipe out the species in an area since that has dramatic effects on the whole ecosystem. The researchers conducted many trials and found that, in a week, a ratio of about “5 or 6 sterile males to one wild-type male” decreased female fertility by 50% while, a ratio of 15:1 suppressed female fertility to about 20%, where it leveled off. So depending on the situation, they now release the more precise amount. 

I think that this is one of the brilliant uses of CRISPR, and it only goes to show how far we can go if we master this technique. An ethics question that this research brings up is, do humans have the right to wipe out an entire species just because it is causing harm to humans?

CRISPR Gene Editing: The Future of Food?

Biology class has taught me a lot about genes and DNA – I know genes code for certain traits, DNA is the code that makes up genes, and that genes are found on chromosomes. I could even tell two parents, with enough information, the probabilities of different eye colors in their children! However, even with all this information, when I first heard “gene editing technology,” I thought, “parents editing what their children will look like,” and while this may be encapsulated in the CRISPR gene editing technology, it is far from its purpose! So, if you’re like me when I first started my CRISPR research, you have a lot to learn! Let’s dive right in!

CRISPR

Firstly, what is CRISPR Gene Editing? It is a genetic engineering technique that “edits genes by precisely cutting DNA and then letting natural DNA repair processes to take over” (http://www.crisprtx.com/gene-editing/crispr-cas9).  Depending on the cut of DNA, three different genetic edits can occur: if a single cut in the DNA is made, a gene can be inactivated; if two separate DNA sites are cut, the middle part of DNA will be deleted, and the separate cuts will join together; and if the same two separate pieces of DNA are cut, but a DNA template is added, the middle part of DNA that would have been deleted can either be corrected or completely replaced. This technology allows for endless possibilities of advancements, from reducing toxic protein to fighting cancer, due to the countless ways it can be applied. Check out this link for some other incredible ways to apply CRISPR technology!

In this blog post however, we will focus on my favorite topic, food! Just a few months ago, the first CRISPR gene-edited food went on the market! In Japan, Sicilian Rouge tomatoes are now being sold after the Tokyo-based company, Sanatech Seed, edited them to contain an increased amount of y-aminobutyric acid (GABA). “GABA is an amino acid and neurotransmitter that blocks impulses between nerve cells in the brain” (https://www.scientificamerican.com/article/crispr-edited-tomatoes-are-supposed-to-help-you-chill-out/). It supposedly (there is scarce scientific evidence of its role as a health supplement) lowers blood pressure and promotes relaxation. In the past, bioengineers have used CRISPR technology to “develop non-browning mushrooms, drought-tolerant soybeans and a host of other creative traits in plants,” but this is the first time the creation is being sold to consumers on the market (https://www.scientificamerican.com/article/crispr-edited-tomatoes-are-supposed-to-help-you-chill-out/)!

Tomatoes

So, how did Sanatech Seed do it? They took the gene editing approach of disabling a gene with the first method described above, making a single cut in the DNA. By doing so, Sanatech’s researchers inactivated the gene that “encodes calmodulin-binding domain (CaMBD)” in order to increase the “activity of the enzyme glutamic acid decarboxylase, which catalyzes the decarboxylation of glutamate to GABA, thus raising levels of the molecule” (https://www.scientificamerican.com/article/crispr-edited-tomatoes-are-supposed-to-help-you-chill-out/). These may seem like big words, but we know from biology that enzymes speed up reactions and decarboxylation is the removal of carbon dioxide from organic acids so you are already familiar with most of the vocabulary! Essentially, bioengineers made a single cut in DNA inside of the GABA shunt (a metabolic pathway) using CRISPR technology. They were therefore able to disable the gene that encodes the protein CaMBD, and by disabling this gene a certain enzyme (glutamic acid decarboxylase) that helps create GABA from glutamate, was stimulated. Thus, more activity of the enzyme that catalyzes the reaction of glutamate to GABA means more GABA! If you are still a little confused, check out this article to read more about how glutamate becomes GABA which will help you better understand this whole process – I know it can be hard to grasp!

After reading all of this research, I am sure you are wondering if you will soon see more CRISPR-edited food come onto the market! The answer is, it depends on where you are asking from! Bioengineered crops are already hard to sell – many countries have regulations against such food and restrictions about what traits can actually be altered in food. Currently, there are some nutritionally enhanced food on the market like soybeans and canola, and many genetically modified organisms (GMOs), but no other genome-edited ones! The US, Brazil, Argentina, and Australia have “repeatedly ruled that genome-edited crops fall outside of its purview” and “Europe has essentially banned genome-edited foods” (https://www.scientificamerican.com/article/crispr-edited-tomatoes-are-supposed-to-help-you-chill-out/). However, if you are in Japan, where the tomatoes are currently being sold, expect to see many more genome edited foods! I know I am now hoping to take a trip to Japan soon!

Thank you so much for reading! If you have any questions, please ask them below!

Forbidden Baby Editing

We all at this point in life have come to know what gene editing is. The technology for it is slowly and forever becoming more and more advanced. The scary thing about editing genes is the fact that we have to potentially affect a baby’s life their entire time alive. It has many different problems which is why its going to take a long time for it to fully get approved in the hospital.

Well unfortunately in an article found here there was a fright to figure out that someone had actually edited the genomes of some babies without people knowing. Many scientists condemned scientist He Jianku as it came to light that he had done something that the science was not ready for yet. He used CRISPR Cas9 tech in order to alter some genes of a few babies. The definition of CRISPR is here but basically it is a general tech to edit the genomes of babies that haven’t been born yet. People were up in arms about the process because he had bypassed the ethical laws and needed up editing the genes of a real live human. People in the science community go on to say that the CRISPR technology just isn’t ready to be executed on a human. There needs to be many more trials before it is used on a person for real. There is progress to make sure this doesn’t happen such as fines and bans from research however they are trying to make sure that it doesn’t happen at all. It gives scientists a bad name and he is trying his best to not let that happen. Technology will always advance and the hard part is trying to make sure that tech is ethical. Hopefully this gives insight to how we can prevent things like this happening in this day and age

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