BioQuakes

AP Biology class blog for discussing current research in Biology

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

Breaking the Chains of Sickle Cell: A New Dawn with Gene Therapy

The U.S. Food and Drug Administration has made a significant advancement in the treatment of sickle cell disease (SCD) by approving two new cell-based gene therapies, Casgevy and Lyfgenia, for patients aged 12 and older. Sickle cell disease is a genetic blood disorder that affects about 100,000 people in the U.S., predominantly African Americans, and is characterized by a mutation in the hemoglobin protein. This mutation leads to red blood cells adopting a crescent shape, which can obstruct blood flow and oxygen delivery, causing severe pain, organ damage, and potentially life-threatening complications.

The mutation in the hemoglobin protein that characterizes sickle cell disease (SCD) alters the structure and function of hemoglobin, which is crucial for transporting oxygen in the blood.  Hemoglobin is made up of four protein subunits, and in SCD, a mutation occurs in the gene that codes for the beta-globin subunit. This mutation leads to the production of an abnormal form of beta-globin known as hemoglobin S (HbS). In normal RBC (red blood cells), hemoglobin (a protein) has a particular shape. We learned in AP biology that proteins need a specific shape to carry out their function. In people with sickle cell anemia, that protein is mutated doesn’t have the correct shape, and cannot carry out its function.  The reason it doesn’t have the right shape is that the mutated hemoglobin sequence is modified at a single amino acid.

Under certain conditions, such as low oxygen levels, dehydration, or acidosis, HbS molecules tend to stick together, forming long, rigid chains within the red blood cells. These chains distort the shape of the red blood cells from their normal, flexible disc shape to a rigid, crescent or “sickle” shape. Unlike normal red blood cells that can easily move through the bloodstream, these sickled cells are stiff and sticky. Its interesting how such a small change can have such a significant effect in our body!

The crescent-shaped cells can get trapped in small blood vessels, blocking the flow of blood. This blockage prevents the delivery of oxygen to nearby tissues, which can cause pain and damage to tissues and organs. Furthermore, the sickled cells are more prone to breaking apart, leading to hemolysis (the destruction of red blood cells), which can cause anemia (a shortage of red blood cells) and other complications. The recurring blockage of blood vessels and the chronic shortage of red blood cells and oxygen supply lead to the severe symptoms and complications associated with sickle cell disease, including acute pain crises, increased risk of infections, and organ damage.

Casgevy stands out as the first therapy of its kind to employ CRISPR/Cas9, a groundbreaking genome editing technology, to modify patients’ hematopoietic stem cells. This process aims to increase the production of fetal hemoglobin in patients, which helps prevent the sickling of red blood cells. On the other hand, Lyfgenia uses a lentiviral vector to genetically modify blood stem cells to produce a variant of hemoglobin that reduces the risk of cells sickling. Both therapies involve modifying the patient’s own blood stem cells and reintroducing them through a one-time infusion, following a high-dose chemotherapy process to prepare the bone marrow for the new cells.

Crispr

These therapies represent a major leap forward in treating sickle cell disease, addressing a significant unmet medical need for more effective and targeted treatments. The FDA’s approval of Casgevy and Lyfgenia is based on the promising results of clinical trials, which demonstrated a substantial reduction in the occurrence of vaso-occlusive crises, a common and painful complication of SCD, among treated patients.

The approval of these therapies also underscores the potential of gene therapy to transform the treatment landscape for rare and severe diseases. By directly addressing the genetic underpinnings of diseases like SCD, gene therapies offer a more precise and potentially long-lasting treatment option compared to conventional approaches. The FDA’s support for such innovative treatments reflects its commitment to advancing the public health by facilitating the development of new and effective therapies.

However, it’s important to note that these therapies come with risks and side effects, such as low blood cell counts, mouth sores, and the potential for hematologic malignancies, particularly with Lyfgenia, which carries a black box warning for this risk. Patients receiving these treatments will be monitored in long-term studies to assess their safety and effectiveness further. Despite these challenges, the approval of Casgevy and Lyfgenia marks a hopeful milestone for individuals with sickle cell disease, offering new avenues for treatment and the promise of improved quality of life. If you were diagnosed with Sickle cell disease, would you try this no-treatment when available? Do the positives outweigh the negatives? Let us know!

Gene Editing Used to Eliminate Invasive Rodent Species’ on Islands

Species of Invasive House Mice have been not just a nuisance, but potentially dangerous and damaging on islands for hundreds of years. These house mice can be dangerous, as they have the potential to spread diseases by getting into food stores or biting humans, to cause asthma or allergy flare ups, and to bring unwanted insects such as fleas, ticks, or  lice into a home. Scientists have been looking for a way to remove these invasive pests from homes throughout time, and to no avail. Now, they have found a new way to eliminate entire populations of these pests at a time in a mere 25 years. 

Mouse white background

With the emergence of DNA editing technology, scientists have found  a way to edit the mice’ DNA so that a certain chunk of the edited DNA is inherited way more often than the average trait. This lab-created trait is called a gene drive, which had in the past been used to successfully reduce many pesky populations of insects before, but had not been proven effective in mammals. To fix this issue, scientists decided that they most discover more about the haplotypes, which are “naturally occurring group(s) of genes that gets passed on as a unit during replication” within house mice. They discovered that the t-haplotype within house mice get passed on to offspring 95% of the time, instead of the usual percentage of 50%. The editing of this t-haplotype was found to be very favorable. This haplotype evolved naturally within these house mice, meaning that will continue to be present in the wild, and there is no projection of resistance to this haploytpe being found anytime soon. Another reason why the editing of this gene sequence is favorable is that it is only present in the invasive species of house mice, meaning that it will not effect other noninvasive species

 

Now the only question is, how will scientists change this haplotype? Well, as CRISPR technology is emerging and evolving, it has been found as the obvious tool to use to edit this gene. Molecular Biologists have used CRISPR to edit the mice’ DNA to add the CRISPR tool into the t-haplotype. There are two affects of this change, when male mice with a heterozygous genotype of the edited gene mate, the CRISPR genes inserted will cause any baby female mice created to be infertile. The other effect of this genetic change is that males with the homozygous genotype of the edited gene will be sterile.

CRISPR logo

Now you might be asking, “has this format gene editing to eliminate the population of the invasive house mice actually been proven effective in any way?” Well, the answer to that is complicated, as scientists have not yet properly tried it out on any island populations. They have used computer simulations to test their hypothesis, finding that in the simulation that after adding 256 mice with the altered gene into the population, the island population of this mouse would go extinct within 25 years. Scientists have still only tested the changing of the t-haplotype within these mice in labs, and have not yet tested the use of CRISPR to effectively damage genes needed for fertility in the house mice. More testing must be done to effectively ensure that this method of eliminating the species is effective, and so we might have to wait some years to begin the overall mission. Overall, scientists are hoping to find a way to eliminate populations of invasive species such as the house mouse in timelines smaller than 25 years,  and many are looking to the future of CRISPR technology as the true way to achieve this goal.

CRISPR gene editing: The Benefits and the risks

CRISPR gene editing is a precise technique that uses the Cas9 enzyme and gRNA to modify DNA sequences in an organism’s genome. This method is inspired by a natural bacterial mechanism that protects against viruses. It can change existing genes, introduce new genetic material, and revolutionize fields such as industry, agriculture, and medicine.

CRISPR gene editing was first invented in 1987 by Ishino Etal. Scientists first hypothesized that prokaryotic cells use this method as part of their adaptive immune systems. However, this method was not elucidated until 2007. This gene-editing technique uses RNA molecules to direct the Cas9 enzyme to the precise location where the DNA strands are being cut, thus allowing genetic materials to be modified or added. To be more specific, this system relies on the enzyme’s ability to cleave DNA double helix strands at a particular location, allowing scientists to modify the DNA sequence. This technique is especially beneficial to the medicinal fields due to its specificity; it can potentially treat genetic diseases such as cystic fibrosis, Alzheimer’s, Huntington’s, Parkinson’s, or cancer by modifying the immune cells and directing them to target and kill cancer cells.

CRISPR-Cas9 Editing of the Genome (26453307604)

Despite the benefits, CRISPR also contains some serious risks. A specific protein called p53, also known as the “guardian of the genome,” helps to detect any damage in the DNA and thus; heads the cells to stop diving to prevent any mistakes. The CRISPR technique might trigger a p53 response, in which edited cells can be “tagged” as damaged and eliminated, thus reducing the efficiency of the gene editing process. However, recent research also indicates that CRISPR can lead to cell toxicity and genome instability. In addition, CRISPR may disrupt normal cell functioning, which leads to cells being unable to detect any DNA damage or extra cell division, thus increasing the risk of further mutations.

Nonetheless, CRISPR still goes deep down into our biology field as it contains molecular biology, where it goes deep down into the cells and modifies DNA sequence. However, changing an organism’s DNA sequence using CRISPR gene-editing technology could have unintended consequences such as off-target effects, incomplete editing, and unknown long-term effects such as cancer or DNA mutation if the matching went wrong.

In First, Scientists Use CRISPR for Personalized Cancer Treatment

Behold, have researchers found a groundbreaking method to fight tumors? Could genome-edited immune cells finally provide a way to defeat cancer?

In a recent clinical trial, immune cells were modified by CRISPR gene editing to recognize mutated proteins specific to tumors. When released into the body, the cells could target and kill the specific tumor cells. This cancer research utilized gene editing and T-cell engineering.

The trial involved 16 individuals who suffered from solid tumors (including breast and colon cancer). The results were published in Nature by Heidi Ledford and then presented on November 10, 2022 in Boston, Massachusetts at The Society for Immunotherapy of Cancer conference. The findings were later released in Scientific American.

According to Antoni Ribas, a co-author of the study and a cancer researcher and physician at the University of California, Los Angeles, ” It is probably the most complicated therapy ever attempted in the clinic.” He describes the process as “trying to make an army out of a participant’s own T cells.”

To begin the study, Ribas and his colleagues ran DNA sequencing on each patient’s blood sample and tumor biopsies. The goal was to identify unique mutations of the timer, but not present in the blood. Ribas notes that these mutations differ across different types of cancer, with only a few being shared. Then using algorithms, Ribas’s team predicted which mutations were the most likely to initiate a response from the T cells(a type of white blood cell that functions to notice and destroy irregular cells); however, immune systems rarely destroy cancerous tumors. With that being said, the team used CRISPR gene editing to insert designated t-cell receptors that recognized the tumor. Patients were given medication to reduce normal immune cells before the researchers infused the engineered cell.

Joseph Fraietta, who specializes in designing T-cell cancer therapies at the University of Pennsylvania in Philadelphia, describes the process as “tremendously complicated”, for some cases could take more than a year to complete in certain cases.

Each individual in the study received T cells engineered to target up to three sites, and after some time, the concentration of the engineered T cells was higher than the average T cells in the bloodstream near the tumors. A month after the treatment, five participants’ tumors had not progressed, and only 2 showed evidence of T-cell activity.

While the treatment’s effectiveness was limited, Ribas notes that a small dose of T cells was used at first and stronger doses would be proven more effective. Fraietta feels “The technology will get better and better.”

Although engineered T cells, also known as CAR T cells, were approved to treat certain blood and lymphatic cancers, CAR T cells only target proteins that are present on the surface of tumor cells, and According to Fraietta, no surface proteins have been discovered in solid tumors. Additionally, tumor cells may suppress immune responses by releasing immune-suppressing chemical signals and consuming local nutrient supplies to promote their rapid growth.

Researchers are hopeful to engineer T cells to not only recognize cancer mutations but also to become more active in the vicinity of the tumor. Potential techniques include ” removing the receptors that respond to immunosuppressive signals, or by tweaking their metabolism so that they can more easily find an energy source in the tumor environment,” as Heidi Ledford, writes in her article. With advances in CRISPR technology, researchers anticipate revolutionary ways of engineering immune cells in the next ten years.

In AP Biology this year, we learn about the Immune system. This topic is specifically related to the adaptive, or pathogen-specific, Immune response. T Lymphocytes, or T cells for short, are a part of the cell-mediated immune response where T-cells can identify, and kill infected or cancerous cells, while also preventing reinfecting.

The Blood Brain Barrier Can’t Block This!

University of Wisconsin-Madison Professor, Shaoqin “Sarah” Gong is ready to take on finding cures for brain disease such as Alzheimer’s and Parkinson’s disease. Gong and her colleagues strive to enable a “noninvasive, safe and efficient delivery of CRISPR genome editors” that can be used as forms of therapy for these diseases. According to MedlinePlus, there are many forms of brain disease, some caused by tumor, injury, genetics; however, Gong’s research focuses on degenerative nerve diseases. Degenerative nerve diseases can affect balance, movement, talking, breathing and heart function. The reason cures for degenerative nerve disease are difficult to create is because of the blood brain barrier. According to the American Society for MicroBiology, the blood brain barrier is a feature of the brain and central nervous system blocking the entrance of “microorganisms, such as bacteria, fungi, viruses or parasites, that may be circulating in the bloodstream”. Unfortunately, the barrier block is a very selective site that won’t let vaccines and therapies through. Fortunately, Gong’s nano-capsules with CRISPR’s genome editors point toward brain disease therapy and a cure.

 

Alzheimer's disease brain comparison

Gong’s study proposes dissolvable nano sized capsules that can carry CRISPR genome editing tools into organs. According to CRISPR Therapeutics, CRISPR technology meaning Clustered Regularly Interspaced Short Palindromic Repeats is an “efficient and versatile gene-editing technology we can harness to modify, delete or correct precise regions of our DNA”. CRISPR edits genes by “precisely cutting DNA and then letting natural DNA repair processes take over.” CRISPR targets mutated segments of DNA that can produce abnormal protein causing diseases such as degenerative nerve disease.  CRISPR works with the help of a guide RNA and Cas9. Together the complex can recognize and bind to a site next to a specific target sequence of DNA that would lead to the production of an abnormal protein. CAS9 can cut the DNA and remove a segment. As a result natural DNA pathways occur and RNA polymerase will return to rebuild and correct the mutated segment. 

via GIPHY

Consequently with the addition of glucose and amino acids the nano-capsules containing CRISPR Technology can pass through the blood brain barrier to conduct gene editing to target the gene for the amyloid precursor protein that is associated with Alzheimer’s. The topic of gene editing coincides with the Gene Expression portion of the AP Biology curriculum. In the topic of gene expressions 2 processes are emphasized: transcription (the process of making an RNA copy of DNA) and translation ( the process of making proteins using genetic information from RNA). In the CRISPR technology the editing of genes closely relates to the process of transcription. Transcription mistakes can be made which can lead to mutations, these mutations can potentially cause nonsense, missense or deletions of nucleotides ultimately producing wrong codons that would code for incorrect/abnormal proteins. However, the CRISPR technology would be able to correct these mutations in the DNA, replacing the incorrect nucleotides to correct ones and preventing the production of abnormal proteins. Fortunately, Gong’s unique nano-capsules have successfully been tested on mice, giving scientists hope that treatments and therapy for these brain diseases are coming soon and can help many.

The End Of Malaria

Introduction

Attention everyone, what if we told you that there is a way to potentially wipe out the bad mosquito species that causes malaria? Scientists have developed a genetic weapon, a self replicating bit of DNA called a gene drive, that interferes with the mosquitoes ability to reproduce. This can be revolutionary and save millions of children’s lives in the future.

What is malaria

Malaria is a deadly disease killing about 643,000 people every year. It is transmitted by a parasite -mosquito bites. The symptoms of malaria include fever, chills, and other flu-like symptoms.

Malaria knocks you flat, keep covered, use your repellent (4647891178)

How it works 

Gene drives work starts with taking one transgenic organism into the lab so it can be modified. It then can be engineered for release into wild populations to spread an altered allele. Two types of drives are possible: modification drives spread an advantageous gene, while suppression drives spread a gene that reduces the population. As the gene spreads this ultimately allows for the death of mosquitoes to spread exponentially. This topic also relates to what we learned in the AP Biology units on genetics and DNA. The connection to genetics is evident in the ability to control breeding of species, such as mosquitoes, using the knowledge of Punnett squares and the principles of dominant and recessive traits. However, the most significant connection between genetics and mosquito control lies in the ability to manipulate and alter DNA.

CRISPR illustration gif animation 1

Future

Gene drives can potentially save millions of lives by reducing mosquito populations and preventing the spread of malaria. The technology is being tested in Africa, where malaria is most prevalent. Soon it will hopefully be around the entire world and save millions of lives all together. 

 

 

CRISPR Tool PASTEs in New Genes

Researchers at Massachusetts Institute of Technology developed a revolutionary new gene editing tool. The tool is called PASTE, and it is a new CRISPR-Cas9 based genome editing tool. It combines traditional CRISPR and integrases, enzymes that can insert or remove DNA sequences, to cut out certain DNA segments and “paste” in other DNA segments. This new method removes the necessity for double-stranded DNA breaks, which can lead to mutations in the DNA sequence. CRISPR logo

PASTE combines CRISPR-Cas9 nickase, which cuts out a singular DNA strand, with serine integrase, an enzyme that can insert a lot of DNA, and reverse transcriptase, an enzyme that allows PASTE to add a single strand of DNA each time while preventing double-stranded DNA breaks. PASTE produces less indels than CRISPR-Cas9 alone. Indels(insertions or deletions are genetic mutations that often occur when a gene is edited. They can alter the function of genes, thus affecting the organism’s overall health or specific traits (New Atlas).

Additionally, PASTE researchers believe that PASTE could possibly treat genetic diseases by replacing “bad” genes with “good” genes. This is because PASTE is great at “pasting” genes into various parts of an organism’s genome. PASTE researchers tested PASTE against homology-independent targeted insertion and homology-directed repair, discovering that paste had higher insertion effectiveness than homology-independent target insertion, but lower insertion effectiveness than homology-directed repair. PASTE, however, produced less “inaccuracies” than homology-directed repair. These inaccuracies occur when the tool inserts DNA into the wrong part of an organism’s genome, effectively risking unwanted effects (Genome Web).

While PASTE is still in its infancy, it is already revolutionizing the gene editing industry. It not only reduces the risk of undesired mutations, but also increases the efficiency of gene insertion. It is pioneering treatment of genetic diseases. 

AP Bio Side Note 🙂

This technology relates to AP Bio because of its use of introns and exons. PASTE can remove or replace introns and exons, depending on what causes the genetic mutation. This is interesting because although introns are noncoding sequences of DNA, mutations in them can still cause negative effects in people. Additionally, while more intuitive, it is also revolutionary that technology is able to replace exons. I am excited to see what the future for Crispr tools holds. Please leave a comment if you found this post interesting!

No need to buy fragrances, we can just create them: a new way of creating everyday items from scratch.

Gene modification.

In a rapidly developing industry, genome editing technology has been growing to a point where “food, drugs, cosmetics, and biofuels” can be synthesized by microbes. Eric Rhodes investigates this phenomenon through the use of CRISPR/Cas9 gene editing technology. Emmanuelle Charpentier and Jennifer Doudna’s findings reveal how scientists can target specific segments in genes and then inactivate, delete, insert, and alter to however the scientist pleases.

At a closer look at what CRISPR technology is, Rhodes, elaborates and shows that multiple genes can be edited. It can be altered to produce any of the approximately 30 biosynthetic gene clusters to produce any natural product. Some popular compounds that are produced include carotenoids, citric acid, 1,3-propanediol, phenylethanol, and squalene. This can make great strides by making common commodities more accessible to the average human. Whether it is pigments (cartenoids), flavoring agents (citric acid), cosmetics (phenylethanol), or components in vaccines (squalene), the opportunities are endless.

We had recently done a bio lab on E.coli and through independent research, we found E.coli’s importance to our digestive system. CRISPR technology too can be used in the engineering of enzymes similar which have seen massive practicality in the modern world. In biology class we learned about epigenetics and how gene expressions can be more pronounced or repressed. In the case of CRISPR technology, CRISPRa involves fusing a catalytically inactive Cas9 (dCas9) protein to a transcriptional activation domain, which can attach transcriptional things to a specific promoter and enhance gene expression.

Perhaps a more pressing matter that this CRISPR technology can target is finding greener alternatives in our world. Rhodes claims that “CRISPR can also be used to modify microbes to grow at lower temperatures” this way high demand species that are endangered will have less pressure of being threatened. This could pose a creative way of solving some endangered species problems by simply providing a cleaner alternative.

The growing potential of genome editing technology, specifically CRISPR/Cas9, to produce a range of useful products from common commodities to components in vaccines, presents endless opportunities for the future of industrial biotechnology, and may help address issues related to endangered species.

CAST is in the past, it’s time for HELIX

CRISPR stands for clustered regularly interspaced short palindromic repeats.’ The term references a series of repetitive patterns in the DNA of bacteria discovered in the 90s. 20 years later, Jennifer Doudna and Emmanuelle Charpentier discovered that CRISPR-Cas9 could be used to cut any desired DNA sequence by just providing it with the right template, meaning it could be used as a gene-editing tool. To add a desired DNA sequence, one needs an upgraded version of CRISPR editing called CAST, CRISPR-associated transposases. Unfortunately,  CASTs suboptimally insert more DNA sequences than wanted and have a relatively high rate of unwanted off-target integration at unintended sites in the genome. This leads to mutations, the three being, silent mutations, missense mutations, and nonsense mutations. A silent mutation is an insertion or deletion of a nucleotide that doesn’t change the amino acid sequence. A missense mutation is an insertion or deletion of a nucleotide that changes one or more of the amino acids. Lastly, a nonsense mutation leads to an early stop signal.

4.2. The CRISPR Cas 9 system II

Luckily, new research published in Nature Biotechnology tells the reader that an improved CAST system called HELIX now exists. Helix stands for Homing Endonuclease-assisted Large-sequence Integrating CAST-compleX. This mouthful dramatically increased the efficiency of correct DNA insertions, reducing insertions at unwanted off-target sites. HELIX has over a 46% increase in on-target integration compared to that of the CAST system. This discovery is one of many that will continue to help us understand the complexity of our genes.

CRISPR: how one tool can change an entire generation (of invasive mice).

In recent years, technology has heavily impacted scientists abilities to change the world. CRISPR is a recently discovered gene editing tool that is revolutionizing the way scientists are treating patients and curing diseases. Recent research has also found that CRISPR can be used to help mice infestations in certain parts of the world (random, but cool)!

CRISPR logoMice infestations are a problem in many islands, and CRISPR is here to help. Scientist believe they had found a way to make an entire mice population extinct (in a few decades) by using gene editing via CRISPR. In order to understand exactly how scientists plan on doing this, it is important to understand what a “haplotype” is. A haplotype is a set of genes that are inherited by the next generation together. The “regular house mouse” has what’s called a “t-haplotype,” and it’s passed down roughly 95% of the time (a lot compared to the normal 50%). The study states that male house mice with two t-haplotype copies become infertile, and females with two t-haplotype copies will become sterile as well. As we know from AP-Biology, when an organism has two copies of some gene, it is known as homozygous- meaning it has two of the same alleles of some gene. In this case the phenotype that makes the mouse homozygous would be the altered t-haplotypes. If a mouse has two of these altered t-haplotype genes, it becomes sterile and cannot reproduce.

Mouse white background

CRISPR plays a crucial role here – by using gene editing through CRISPR technology, scientists are able to edit the t-haplotype of the M. musculus house mouse so that next time a male M. musculus mates with a female, the offspring will become infertile. That’s right, CRIPSR can be used to completely alter and wipe an an entire M. musculus population over the course of a few years. By using computer technology, scientists predict that by adding just 256 “altered” mice to a certain island population of mice, an island of 200,000 mice can be fully wiped out within about 25 years.

Researchers in laboratoryScientists are hopeful, optimistic, and invested in CRISPR technology. The “25 years later” prediction is a long time to wait, and scientists hope that sometime in the future, CRISPR will be able to work faster, allowing problems to be solved more quickly and more efficiently. I think that this study is an important part of CRISPR potential, and it makes me very curious to see what CRISPR has in store for the future, and what other kinds of animal related issues it can help solve.

We Live In a Time Where We Can Hack and Edit The DNA In Diseases, and We Have Only Just Begun…

CRISPR gene editing, (Clustered Regularly Interspaced Short Palindromic Repeats), is a relatively new biological technology that allows scientists to fix unimaginable flaws with an unprecedented minimal risk of off-target effects. This advanced technology aligns perfectly with our current unit of DNA replication and Gene expression/replication, so it should be a good review to keep reading.

CRISPR gene editing has two main components; the Cas9 protein and a guide RNA (gRNA). The Cas9 protein acts as the Helicase, cutting and unzipping the DNA strand. The gRNA is designed to recognize a specific sequence in the DNA of a cell. Once the DNA is cut, the cell uses a homologous DNA template to repair the break in the DNA molecule. The template DNA, Homology Directed Repair (HDR), is designed to carry the desired genetic modification and incorporate it into the DNA through the natural DNA repair mechanisms. This process alters or even adds new genetic information to the organism. For example, researchers from UT Southwestern Medical Center used CRISPR to treat Duchenne muscular dystrophy (DMD). A genetic disease that causes muscle degeneration and weakness. The team used CRISPR to delete the gene responsible for producing a toxic protein that causes DMD. They then replaced the missing gene with a shorter, functional version, which allowed the muscles to regenerate and become stronger.

CRISPR illustration gif animation 1

This advanced technology has been used to increase crop yield from various crops. In California it was used to create more drought resistant rice. In another state, it was used to eliminate browning of red apples. This process is becoming increasingly useful and popular because of its safety. The gRNA can be designed to target a very specific sequence of DNA, which means that scientists can modify genes with precision and accuracy. This specificity also reduces unintended genes, which remains to be a large concern for other gene-editing processes. This technology has enormous potential in the science world and can safely guide us into disease treatments, agricultural efficiency, and advanced biological research.

DCas SAM system

Crispr Gene Editing Aids in Sustainable Bioenergy Production

CAS 4qyz

CRISPR/Cas-9 is the most precise gene editing tool. It is a specific, efficient and versatile gene-editing technology used to modify, delete or correct precise regions in our DNA.

Miscanthus sinensis ja01

New research shows that for the first time, researchers have successfully demonstrated precision gene editing in miscanthus. Miscanthus is a promising crop for sustainable bioenergy production due to its high yield and superior environmental adaptability.

A study was done by the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), a Bioenergy Research Center (BRC) funded by the U. S. Department of Energy. In this study they edited the genomes of three miscanthus species using CRISPR/Cas9.

In this study researchers demonstrated gene-editing in three species of miscanthus: Miscanthus x giganteus, M. sacchariflorus, and M. sinensis. These plants are paleo-polyploids which refer to ancient genome duplications which occurred at least several million years ago. Since these plants are paleo-polyploids the design of the RNAs that locate genetic material for editing needed to target all copies of a gene.

The researchers used the information from what they know about miscanthus and identified RNAs that could target homoeologs, or duplicated gene copies, of the phenotypes in miscanthus plant tissue. To identify miscanthus lines that transformed well, the researchers screened germplasm from commercial vendors and others that help research the study.

Prior to this study, the bioengineering work was limited to sorghum and cane because the methods for precise engineering in miscanthus had not been developed.

This relates to AP biology because CRISPR/Cas 9 technology allows scientists to edit genes and manipulate gene expression with a level of ease that was no possible using other methods. In AP biology we learned how gene expression works and what happens when the encoded gene is changed. Gene expression is accomplished in two main steps: transcription and translation. In transcription an RNA transcript is created from one strand of template DNA. In this stage RNA polymerase binds to prometer, DNA unwinds, polymerase initiates RNA synthesis. The polymerase then moves downstream, unwinding DNA and adding RNA nucleotides. The RNA transcript is then released and RNA polymerase detaches. After transcription translation occurs where cells make proteins using the genetic information carried by the mRNA. This is different in the use of CRISPR/Cas 9 as, unlike coding DNA which gets transcribed and eventually translated into proteins, these regions gets transcribed but never translated.

CRISPR Technology leads the way for potential breakthrough in cancer treatment

According to The American Cancer Society, scientists can alter the structure of a particular white blood cell known as the T-cell.  This method, known as CAR T-cell therapy, has long been established as a potential weapon against cancer, altering T-cells to best fight cancer based on the patient’s own characteristics.  According to an article in Forbes, the genetic editing procedure that has been used to facilitate this technology has relied upon “Viral Vectors,” which according to Beckman Coulter, viral vectors are modified viruses “that can be used to deliver nucleic acids into the genetic makeup of cells.”  While useful, Forbes asserts that the usage of Viral Vectors can be time-consuming and “can cost up to $50,000 per dose.”  For these reasons, scientists have looked towards a new technology, known as CRISPR technology to facilitate the editing of T-cells.CRISPR logo

 

According to the National Human Genome Research Institute, “CRISPR (short for “clustered regulatory interspaced short palindromic repeats”) is a technology that research scientists use to selectively modify the DNA of living organisms.”  According to Forbes, this technology differs from viral vector technology in that it involves the synthesis of “RNA guides,” which allow the scientists to break a DNA sequence at a targeted point, allowing for a change, as would be required to facilitate CAR T-cell therapy.  Furthermore, the article asserts that “synthesizing an RNA guide is cheaper and more efficient than cultivating retroviral vectors,” potentially allowing for the treatment to be more widespread.  As stated in the Forbes article by William A. Haseltine, former professor at Harvard University, “there is potential to propel CAR T design forward by integrating contemporary innovations such as CRISPR/Cas9 technology.”  It is therefore clear that the usage of CRISPR technology for CAR T-cell therapy could revolutionize cancer treatment

 

 

Many of the concepts referenced in this post involve concepts we have learned in AP bio class.  For example, in the immune system section of the cell communication unit, we learned about the various types of T-cells.  For example, we learned how T-killer cells kill infected cells, such as cancer cells, T-memory cells retain information to prevent further infection, and T-helper cells stimulate other T-cells.  From here, we learned how T-cells, more specifically T-killer cells, can be used to fight cancer, which connects to CAR T-cell therapy’s usage of the cells for gene editing. 

 

While CRISPR technology’s use in CAR T-cell therapy is exciting, according to Haseltine, it “still has room for improvement.”  This technology is not fully developed, and will probably need years to be widespread.  But still, the complete implementation of CRISPR technology in CAR T-cell therapy remains an exciting prospect.

 Cancer Detection Using CRISPR Gene Editing

Currently, many are accustomed to invasive cancer diagnostic methods such as endoscopies, colonoscopies, and mammograms. Driven by the desire to discover new methods, a group of researchers from the American Cancer Society developed an alternative method, which is a significant contribution to cancer detection.

Utilizing CRISPR gene editing as their approach, the group of ACS researchers developed an easy-to-use mechanism for detecting small amounts of cancer in plasma. CRISPR gene editing is a method that scientists and researchers have been using to modify an organism’s DNA. CRISPR gene editing is often done for numerous reasons, such as adding or removing genetic material, creating immune defense systems, and repairing DNA. Their detection method also allows healthcare professionals in diagnostics to decipher between malignant and benign cancer-related molecules that they may discover.

CRISPR Gene-Editing

The first step that the researchers made to develop this approach was to design a CRISPR system that creates a manufactured exosome out of two reporter molecule fragments, which they cut. An exosome is a small vesicle that carries material such as lipids, proteins, and nucleic acids after branching out from a host cell. Exosomes are typically involved in detecting cancerous cells because they provide a glimpse into the host cell they branched out from. Therefore, cancerous cells are shown in their exosomes through biomarkers, like micro RNAs (miRNA). In AP Biology class, microRNAs are described as materials that bind to complementary mRNAs to prevent the translation from occurring. MiRNAs are a recent discovery, identified in 1993. It is now concluded that most gene expression is influenced by them, so the researchers made efficient use of miRNA in their experiment. The two fragments of the reporter molecule came together and interacted with the CRISPR’s materials.

Micro RNA Sequence

The researchers concluded that if the targeted miRNA sequence was evident in the combination, the CRISPR system they made would become activated and cut apart the reporter molecule. The researchers specifically targeted miRNA-21, which is often involved in cancer development. The researchers were able to detect miRNA within a combination of similar sequences and later tested their method on a group of healthy exosomes and cancerous exosomes. Their CRISPR system successfully differentiated between the healthy and cancerous exosomes, which makes this system effective for cancer detection. The researchers are confident that their CRISPR gene editing approach to cancer detection will make diagnosis easier on patients and a more efficient process overall.

 

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!

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