BioQuakes

AP Biology class blog for discussing current research in Biology

Tag: #CRISPR/Cas9

More CRISPR Improvements

Crispr-Cas9 is a genome editing tool that is creating a whole lot of buzz in the science world. It is the newest faster, cheaper and more accurate way of editing DNA.  Crispr- Cas9 also has a wide range of potential applications. It is a unique technology that enables geneticists and medical researchers to edit parts of the genome by cutting out, replacing or adding parts to the DNA sequence.  The CRISPR-Cas9 system consists of two key molecules that introduce a mutation into the DNA. The first Molecule is an enzyme called Cas9. Cas9 acts as a pair of scissors that can cut the two strands of DNA at a specific location in the genome so that bits of DNA can be added or removed.  The second is a piece of RNA called guide RNA or gRNA. This consists of a small piece of pre-designed RNA sequence located within a longer RNA scaffold. The scaffold part binds to DNA and the pre-designed sequence guides Cas9 to the right part of the genome. This makes sure that the Cas9 enzyme cuts at the right point in the genome.Screen Shot 2016-04-10 at 4.50.55 PM

CRISPR-Cas9 is efficient compared to previous gene-editing techniques, but there’s still plenty of room for improvement. CRISPR is less efficient when employing the cellular process of homology-directed DNA repair, or HDR, as opposed to nonhomologous end joining.  Jacob Corn, the scientific director of the Innovative Genomics Initiative at the University of California, Berkeley, and his colleagues have come up with a way to improve the success rate of homology-directed repair following CRISPR-Cas9. “We have found that Cas9-mediated HDR frequencies can be increased by rationally designing the orientation, polarity and length of the donor ssDNA to match the properties of the Cas9-DNA complex,” the researchers wrote in their paper, “We also found that these donor designs, when paired with tiled catalytically inactive dCas9 molecules, can stimulate HDR to approximately 1%, almost 50-fold greater than donor alone.”

“Our data indicate that Cas9 breaks could be different at a molecular level from breaks generated by other targeted nucleases, such as TALENS and zinc-finger nucleases, which suggests that strategies like the ones we are using can give you more efficient repair of Cas9 breaks,” coauthor Christopher Richardson, a postdoc in Corn’s lab, said in a statement.

Original Article:

http://www.the-scientist.com/?articles.view/articleNo/45159/title/More-CRISPR-Improvements/

Other Addtional Helpful Links:

http://www.yourgenome.org/facts/what-is-crispr-cas9

 

HIV Resistance to CRISPR/Cas9

A recent study, described in the Science Daily, shows that researches who used the CRISPR/Cas9 to mutate HIV-1 within cellular DNA found that the mutation led to unexpected resistance.

When HIV enters a cell, its RNA genome is converted into DNA and becomes intertwined with the cellular DNA. So the goal for the CRISPR/Cas9 is to target a DNA sequence and cleave viral DNA. The problem is HIV is too good at surviving and thriving despite new mutations, making it more difficult for the CRISPR/CAS9 to target.

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Photo Source

Chen Liang, Senior Investigator at the Lady Davis Institute at the Jewish General Hospital, noted that when they sequenced the viral RNA of escaped HIV, they were surprised to see that majority of the mutations the virus had, instead of resulting from the errors of viral reverse transcriptase, were rather introduced by the cellular non-homologous end joining machinery when repairing the broken DNA.

The mutations to the sequences caused by the HIV were unrecognizable to the Cas9. Thus the resistant viruses just continued to replicate.

This study serves as a cautionary tale for scientists hoping to apply CRISPR/Cas9 as an antiviral. Liang does not believe these efforts are useless, however, as he is hopeful about strategies that could overcome this roadblock. One such strategy would be to target multiple sites with CRISPR/Cas9 or use other enzymes besides Cas9. After the solution is identified, the next step will be figuring out ways to deliver the treatment to patients. Liang is confident that CRISPR/Cas9 will open doors for finding a cure for HIV-1 and many other viruses.

More Info:

http://www.genengnews.com/gen-news-highlights/hitting-hiv-with-crispr-cas9-can-arouse-resistance/81252590/

http://www.techtimes.com/articles/148378/20160409/crispr-cas9-gene-editing-is-not-good-enough-to-beat-hiv-whats-next-in-humanitys-fight-against-the-deadly-disease.htm

 

 

How CRISPR/Cas9 could one day prevent AIDS

CRISPR/Cas9 is a new gene editing tool that can target and modify DNA with great accuracy.  This new tool has many scientific uses, including treatment of many diseases.  Recently, several breakthroughs have been made in treating HIV with CRISPR Cas9.  However, a number of issues with the tool have come up at the same time.

To understand how CRISPR eliminates HIV, one must know how HIV replicates. HIV replicates by taking over a host cell and injecting its RNA into the cell.  This RNA becomes DNA and joins together with parts of the host cell’s DNA.  After entering the cells, the virus can lay dormant for several years, but will eventually start replicating and taking over other cells.  The standard form of treatment for HIV is an antiretroviral.  While antiretrovirals can be very effective at limiting the spread of the disease, it cannot fully remove it or stop it forever.

HIV virus

image source: http://bit.ly/1S4bcWY

The CRISPR Cas9 could potentially be used to inhibit the spread of HIV by editing the virus out of a cell’s DNA.  Researchers at The University of Massachusetts Medical School have been developing a technology to perform this impressive task.  While there have been several successful trials in preventing HIV from spreading, several trials have lead to increased resistance for the HIV.

“When we sequence the viral RNA of escaped HIV, the surprise is that the majority of the mutations that the virus has are nicely aligned at the site where Cas9 cleaves the DNA, which immediately indicates that these mutations, instead of resulting from the errors of viral reverse transcriptase, are rather introduced by the cellular non-homologous end joining machinery when repairing the broken DNA,” says Chen Liang, a senior investigator at the Lady Davis Institute at the Jewish General Hospital and the Associate Professor of Medicine at the McGill University AIDS Centre.

These mutations alter the strand of DNA, preventing the CRISPR Cas9 from recognizing it.  If the CRISPR Cas9 cannot recognize the virus, it cant remove the viral DNA, allowing the virus to create more copies of itself.  Despite these limitations, researchers like Liang are confident that they can succeed.

article: http://esciencenews.com/articles/2016/04/08/hiv.can.develop.resistance.crisprcas9

CRISPR/Cas9 Provides Promising Treatment for Duchenne Muscular Dystrophy

There are nine kinds of muscular dystrophy and of these, Duchenne MD is the most common severe form of childhood MD. It affects about 1 in 5000 newborn males, only in very rare cases has it affected females. DMD is a genetic disorder that causes progressive muscle degeneration and weakness. Patients usually die by age 30 to 40.

DMD is caused by the absence of a protein, dystrophin, that helps keep muscle cells intact. In 1986 it was discovered that there was a gene on the X chromosome that, when mutated, lead to DMD. Later, researchers discovered that the protein associated with this gene was dystrophin. From this information, we can tell that this disorder is sex-linked, which explains why women are mainly carriers.

No one has found an absolute cure for this genetic disorder until now. Even in recent years, people have discovered treatments that will make patients’ lives more bearable, but never reverse the disorder. As a result of these advances, mostly in cardiac and respiratory care, patients are able to live past teen year and as long as in to their fifties, though this is rare. Although there are still drugs being tested like Vamorolone (a “dissociative steroid,” is an anti-inflammatory compound), more treatments on the molecular level are now being considered. However, thanks to recent discoveries and research with the new genetic technology, CRISPR/ Cas9, scientists may have found a treatment for DMD.

This new approach to gene correction by genome editing has shown promise in studies recently. This particular correction can be achieved in a couple ways: one is by skipping exon 51 of the DMD gene using eterplirsen (a morpholino-based oligonucleotide). Studies over four years show prolonged movement abilities, and a change in the rate of decline compared to controls. The newest approach to gene correction using CRISPR/Cas9, which the article I’m writing about focuses on, was performed in this study as next described: the CRISPR/Cas9 system targets the point mutation in exon 23 of the mdx mouse that creates a premature stop codon and serves as a representative model of DMD. Multiple studies in three separate laboratories have provided a path and laid the groundwork for clinical translation addressing many of the critical questions that have been raised regarding this system. The labs also discovered by further demonstrations, that this is a feasible treatment for humans. Functional recovery was demonstrated in the mice, including grip strength, and improved force generation- all of which are very important and hopeful discoveries. It is estimated from these studies that this new method will pass clinical trials and go on to benefit as many as 80% of DMD sufferers. Even greater success rates are expected if this is performed in young and newborn DMD patients.

A Cure to HIV is Near, But Not Here Yet

The study of genetics, specifically gene editing, has taken monumental leaps over the past few years. One of the biggest achievements of late is the discovery and further research into CRISPR/Cas9. Being able to use CRISPR/Cas9 to edit the genome sequences of living cells far has been the efficient tool geneticists have dreamed of. However, a recent study proved that CRISPR/Cas9 is not yet able to work as the perfect antiviral mechanism.

Image courtesy of AJC ajcann.wordpress.com, https://flic.kr/p/c9ktfQ

Image courtesy of AJC ajcann.wordpress.com, https://flic.kr/p/c9ktfQ

Scientists from McGill University, the University of Montreal, the Chinese Academy of Medical Sciences and Peking Union Medical College did a study where CRISPR/Cas9 was inserted to the replicative process of the HIV invested cell. After HIV enters a cell it’s RNA is converted to DNA which attaches to a cell’s pre-existing strand of DNA. This is when CRISPR/Cas9 is used, it breaks up these two DNA strands. The study found that many of the targeted viruses were killed, however the others viruses developed mutations on even just one nucleotide that made them more resistant and impossible for Cas9 to identify. In conclusion, scientists realize they may need to target more than one region of the DNA at once to effectively kill viruses like HIV.

This topic is very interesting to me because it reflects how we are on the cusp of some incredible biological achievements. I am particularly interested in this study because the effect of HIV/AIDs has devastated not only our country, but also the world, and this study seems like an important step in finding the cure that could save millions of lives. CRISPR/Cas9 seems to offer amazing possibilities, and this is one specific area that grabbed my attention. Do you think a solution to currently incurable diseases is near? Why/Why not? Let me know in the comments below.

Sources:

https://www.sciencedaily.com/releases/2016/04/160407132307.htm

 

Crispr 9, A Dangerous New Field

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Crispr 9 Editing

With the new developments in gene altering, scientists have begun to use technology to alter the gene sequence of embryos. According to an article by Tia Ghost, Chinese scientists have modified the genes of human embryos with mixed results. The idea behind the research was that they would cut out a faulty gene in the DNA sequence and replace it with a correct one, therefore improving the embryo. This is done through a stretch of RNA called CRISPR targets places on the genome that are then cut by Cas9, an enzyme that cuts out specific strands of DNA leaving a spot to be filled within the genome. Scientists then provide a new strand of DNA as replacement. This method is effective in all different kinds of animals as well as humans.

However, the technology is not yet accurate enough to become common practice. According to a leading scientist in the field “the CRISPR technology is simply too risky to use in embryos” at this point. The issue arises in the fact that the RNA sometimes goes to a different site then the one desired, slicing out a necessary part of the genome and replacing it with useless information. This could lead to harmful mutations in the embryo, the opposite of what the scientists want. Even if the technology was at a higher level, editing embryos is still a large ethical dilemma. Some scientists feel that they should not alter life, but simply let it play out the same way it has for billions of years. Other’s argue that each child deserves the best possible chance they can get. Both have strong arguments, and only time will tell which side will win out.

 

 

 

Original Article:

http://www.livescience.com/50596-what-are-genome-editing-risks.html

Biomedical Engineers paving the way for Immunology

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

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

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

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

CRISPR stands for Clustered Regulatory Interspaced Short Palindromic Repeats

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

koli-bacteria-123081_960_720

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

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

ORIGINAL Article: https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology

What came first, the chicken, the egg, or the allergic reaction?

A new study showed the beneficial effects CRISPR/Cas9 can have on those with allergies… in this case, to chickens! For those who don’t know, CRISPR/Cas9 is a gene-editing tool that is used to target certain parts of DNA and modify, disable or enable them. The tool haScreen Shot 2016-04-11 at 12.45.11 AMs been used all across science to inhibit diseases, fix problems with fetuses, change traits, and now to help genetically modify food. Using CRISPR/Cas9 is different than the current definition of genetically modified, which includes injecting chemicals into the food to maximize the amount or change some part of it. This means we humans are ingesting the chemicals; this has led to many concerns. However, CRISPR/Cas9 uses a different approach.

In this specific example, CRISPR/Cas9 creates knockout chickens, or chickens that have had their genes “knocked out”, turned off. Specifically, the ovalbumin (OVA) and the ovomucoid (OVM) genes.  These genes code for proteins that are found in egg whites. It has been discovered that many people are allergic to the proteins produced, so CRISPR/Cas9 targets the genes and turns them off and no proteins are produced. These “genetically modified” eggs are the same as regular eggs just hypoallergenic. In addition, some vaccines are made with egg whites, CRISPR/Cas9 will make it possible for the people who usually have an immune response to the egg whites in those vaccines, to safely receive them. One of the most notable vaccines that uses egg whites is influenza, a very popular vaccine that most of the population receives, and those who couldn’t were at a disadvantage before CRISPR/Cas9. The scientists have said they will continue to cross the modified chickens to see if they are able to knockout more common allergens. So no matter if the chicken or the egg came first, they are now both safe to consume by humans.

 

Evolution: 1, Humans: 0 – HIV Virus has evolved to evade latest gene-editing treatments.

The Human Immunodeficiency Virus (HIV) is notorious for its rapid evolution and elusiveness to our treatments.  Our latest attempt to beat it has been foiled, yet again. As explained in this article, researchers have attempted to eliminate the virus through a genome editing technique called CRISPR-Cas9.  This technique allows scientists to target a specific genetic sequence in a cell to cut, using the Cas9 enzyme and a guiding sequence, and change the function of the gene by inserting corrected/modified sequences.

HIV budding from a Lymphocyte http://https://en.wikipedia.org/wiki/HIV/AIDS#/media/File:HIV-budding-Color.jpg

HIV budding from a Lymphocyte
[Picture Source Link]

This highly versatile technique was recently applied to HIV, in an attempt to disable it and prevent further infection from it.  The technique would theoretically delete HIV genetic sequences in an infected cell and prevent further virus production; however, a recent study  shows that the virus evolves rapidly to avoid this treatment.  The fault lies in the fact that the gene-editing technique targets a specific locus on the DNA to modify.  The treatment was successful in destroying HIV genes in that area of the DNA, but the cell’s repair mechanisms allowed the removed HIV genes to be repaired with new sequences.  This means that the new HIV genes will not be targeted by the CRISPR mechanism, because it contains a different marker, and the virus will live long enough to reproduce.  This rapid microevolution demonstrates the power of natural selection: a predator destroys the majority of the population, but those that are adapted to survive the conditions will live long enough to reproduce and pass on its traits! HIV has eluded us once again, but we now know that the gene-editing CRISPR-Cas9 system will work, provided that we don’t miss any HIV loci…

The research looks promising, but will this be our golden ticket?

 

Original Article: “CRISPR/Cas9 Gene Editing Is Not Good Enough To Beat HIV: What’s Next In Humanity’s Fight Against The Deadly Disease” (Tech Times)

Original Study & Further Reading: “CRISPR/Cas9-Derived Mutations Both Inhibit HIV-1 Replication and Accelerate Viral Escape” (Cell Reports Journal)

Image Source: Wikimedia Commons

 

CRISPR Inhibited by Nucleosomes

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

File:Nucleosome 1KX5 colour coded.png

Structure of a Nucleosome

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

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

Original Article

Forget DNA, Let’s Talk RNA!

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Photo of RNA (licensing information here)

The genetic code within DNA is responsible for determining who we are and what we are capable of. Because of this, scientists have been interested in cracking the genetic code and finding ways to alter it. There are many diseases linked to DNA, as well as RNA. However, scientists have not been as successful in targeting RNA in living cells as they have been in targeting DNA. Recently, using CRISPR-Cas9, researchers at University of California, San Diego School of Medicine have figured out how to do what has been troubling scientists.

Senior author Dr. Gene Yeo described how the researchers at UCSD have been tracking the movement of RNA throughout cells and plan to measure other RNA features and help to correct disease-causing RNA behaviors using CRISPR-Cas9. The location of RNA in a cell determines whether proteins are produced at the right time and in the right place. When defective RNA transport occurs, diseases ranging from autism to cancer can occur. In order to successfully treat these conditions, researchers must find a way to track and measure the movement of RNA. This process was first seen with DNA: scientists found they could use CRISPR-Cas9 to track and edit genes in mammalian systems. Now, however, Yeo and his colleagues at UC Berkeley have started to target RNA in live cells (RNA-targeted Cas9 or RCas9), as well as DNA in live cells.

When CRISPR-Cas9 is used for normal DNA-involved purposes, researchers design “guide” RNA to match the DNA sequence of the gene Cas9 is targeting. The “guide” RNA then directs the Cas9 enzyme to the target spot in the genome. The Cas9 enzyme then cuts the DNA, which causes the DNA to break in a manner that inactivates the gene. Researchers can also replace the section of the genome next to the cut DNA with a corrected version of the gene. In order to allow Cas9 to work for RNA as well as DNA, work originated by co-author Dr. Jennifer Doudna at UC Berkeley laid a base foundation for researchers to design the PAMmer: a short nucleic acid. The PAMmer works with the “guide” RNA to direct Cas9 to an RNA molecule, instead of DNA.

All in all, CRISPR-Cas9 is responsible for a revolution in genomics with it’s ability to target and modify human DNA. Although this breakthrough is crucial, scientists are now trying to use their lead to target and modify RNA. With an extension on already existing research, there is no doubt that scientists will soon be able to do more than just track RNA. So, let’s forget about DNA and shine a light on RNA for a little while!

Source

Harmless Mosquitoes…Yes Please

What are the most annoying things on Earth? Why, mosquitoes of course. They bite you and their bites are extremely irritating. Mosquitoes also carry life-threatening viruses, such as Malaria. However, scientists have come up with a way to get rid of mosquitoes carrying Malaria with the help of gene drives.

A gene drive is a self-generating “cut-and-paste system” that can sterilize mosquitoes. Well how do gene drives work? They operate using CRISPR/Cas9, precision molecular scissors that cut DNA. Scientists used CRISPR/Cas9 to disrupt the genes that are active in mosquito ovaries. If a female mosquito is missing one of these genes, they become sterile. Gene drives insert themselves into a target gene to assimilate every unaltered gene they pass. They break normal inheritance rules by being able to pass themselves into over 50% of an altered animal’s offspring.

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The first gene drive that was made stopped mosquitoes from transmitting Malaria. This new gene drive would eliminate Malaria-carrying mosquitoes in the future by making the females sterile, unable to reproduce. This gene drive is not 100% perfect yet, but scientists are hoping to perfect it soon to be able to release it. They hope that this gene drive will be able to control different insect populations, not only mosquitoes.

Source Article

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