CRISPR/Cas9 is in clinical trials to be used as a therapeutic agent for genetic diseases.
CRISPR stands for “clustered, regularly interspaced short palindromic repeats”, which refers to a DNA region in prokaryotes that contains snippets of viral DNA. The repeats are markers placed between the spacers, and the spacers are the snippets of viral DNA. Bacteria and archea use CRISPR-Cas as one of the defense mechanisms against phages (viruses that infect these prokaryotes).
The CRISPR locus has spacers, repeats, and cas genes. A locus in genetics is a location on a chromosome. Gene mapping is the process of determining the locus for a particular biological trait. In this case the biological trait is the CRISPR immune system, which protects a prokaryote from phages or plasmids.
CAS/CSE protein family includes the mammalian cellular apoptosis susceptibility (CAS) proteins and the yeast chromosome-segregation protein, CSE1. CAS is involved in both cellular apoptosis and proliferation. 1
CRISPR technology can be used to edit DNA in live cells. Different methods can be used to introduce the RNA and DNA-breaking enzyme into the host, including viruses and DNA Transfection 2 . Once the virus injects the payload, the short RNA guides a nuclease to the host DNA. Nuclease is an enzyme that can cleave a chain of nucleotides at the phosphodiester bond.
A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been modified to edit genomes, instead of its original function of attacking viral DNA. The Cas enzyme is guided by a copy of the spacer RNA. To edit the genome, the guide RNA is a section of the genome instead of viral DNA. Within a prokaryote, the CRISPR/Cas machinery is always a danger to itself. If by chance the enzyme responsible for adding new viral DNA, as spacers, gets hold of DNA that matched the prokaryote’s own DNA, then the same CRISPR/Cas machinery would be used in the destruction of the endogenous DNA.
When CRISPR technology is used on embryos, there is a goal to change all the genome in all the cells, rather than create a being with a DNA mosaic. 3
The CRISPR/Cas9 system is not the first technology, but it is more accurate, less costly, and faster than the protein based targeting that has been used previously.
By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's DNA can be cut at a desired location, allowing existing genes to be removed and/or new ones added, through the cell's own DNA repair mechanisms, such as homologous recombination. There is a risk that the homologous recombination will not be perfect and create a defect, or mutation. There is also a risk that the guide RNA will attach to the wrong place on the host DNA 4
“ CRISPR (clustered regularly interspaced palindromic repeats) is a defense mechanism, present in bacteria and archaea, which confers immunity against phages. All species of bacteria and archaea are parasitized by viruses known as phages. Accordingly, prokaryotes have evolved many different types of protection against infection by phages and other sources of foreign DNA. These include prevention of absorption, blocking of injection, abortive infection, and the restriction-modification system (Horvath and Barrangou. 2010). CRISPR DNA sequences and their associated proteins are one such type of protection. The CRISPR system protects prokaryotic cells by destroying viral DNA after it has entered the cell.
Phages infect prokaryotic cells by binding to surface proteins, injecting their DNA through the cell wall, and hijacking the cell’s protein machinery to replicate the DNA. If, for any reason, the DNA is destroyed before it can cause infection, small fragments (21-72 bp) are integrated into dedicated loci (called CRISPR loci) within the cell’s genome. Later, if the cell encounters foreign DNA, it will compare it to the short stored sequences. If the sequences of the stored and foreign DNA match, the foreign DNA will be destroyed by enzymes (Horvath and Barrangou. 2010).
The CRISPR system’s ability to precisely and reliably cleave DNA has made it an active area of study for purposes of genetic engineering ”
Quotes from: CRISPR Immune System in Baceria and Archea
“ Viruses outnumber prokaryotes by ten to one and are said to kill half of the world's bacteria every two days. Prokaryotes also swap scraps of DNA called plasmids, which can be parasitic — draining resources from their host and forcing it to self-destruct if it tries to expel its molecular hitch-hiker. It seems as if nowhere is safe: from soil to sea to the most inhospitable places on the planet, genetic invaders are present.
Prokaryotes have evolved a slew of weapons to cope with these threats. Restriction enzymes, for example, are proteins that cut DNA at or near a specific sequence. But these defences are blunt. Each enzyme is programmed to recognize certain sequences, and a microbe is protected only if it has a copy of the right gene. CRISPR–Cas is more dynamic. It adapts to and remembers specific genetic invaders in a similar way to how human antibodies provide long-term immunity after an infection. ”
“ Mojica and others deduced the function of CRISPR–Cas when they saw that DNA in the spaces between CRISPR's palindromic repeats sometimes matches sequences in viral genomes. Since then, researchers have worked out that certain CRISPR-associated (Cas) proteins add these spacer sequences to the genome after bacteria and archaea are exposed to specific viruses or plasmids. RNA made from those spacers directs other Cas proteins to chew up any invading DNA or RNA that matches the sequence. ”
Quote from: CRISPR origins
“ CAS/CSE protein family includes the mammalian cellular apoptosis susceptibility (CAS) proteins and the yeast chromosome-segregation protein, CSE1. CAS is involved in both cellular apoptosis and proliferation. ” 1
ASSIGNMENT: Dr. Metcalf discusses the idea that the technologies that are used to make GMOs use molecules and processes that were discovered in and borrowed from nature. Consider your views; look up a recent news item on GMO bacteria, yeast, plants, or animals that mentions a peer-reviewed research article. Write a post that addresses the following: Did the content of this lesson change your understanding of GMOs? Why or why not? Do you feel the same or different about GMO microbes, such as bacteria or yeast, and GMO plants? Summarize the news article you have identified in a few sentences. In the article you found, what gene or genes were introduced into what organism? What was the goal? What are potential benefits and risks? How clearly does the article describe the biology of the research conducted?
Dr. Metcalf states that:
“ horizontal gene transfer is rampant in nature. In fact, microbes have evolved systems to allow them to transfer genes between highly unrelated organisms. So for example, the common lab bacterium E.Coli has a piece of DNA that encodes what I called transfer functions. These are designed to transfer DNA from one microorganism to another, to allow them to share their genetic capabilities. E.Coli can do this DNA transfer from one E.Coli to another E.Coli. Or from one E.Coli to a bacterium that it's not really very closely related to or to a fungus or to a plant cell or in fact even to human cells. The efficiency with which this occurs is fairly low but it happens in nature all the time. And as we have begun to amass a collection of genome sequences Tens of thousands of bacterial genome sequences. Thousands of plant and animal genome sequences and we begin to look at those genes and each gene has encoded within it kind of a genetic history. We can look at where the genes origin comes from. We now realize that somewhere between 5 and 20% of any genome including the human genome, comes from unrelated organisms that we got through horizontal of genes transfer. So in fact, horizontal gene transfer. Genetic modification of organisms. By taking genes from one organism and putting them in an unrelated organism happens all the time in nature. It's not uncommon, in fact, it's so common as to be the rule. When we sequence a new microbial genome we know that 10 to 20% of the genes are going to be there by horizontal gene transfer from distant relatives, pieces of DNA that they picked up from the environment or that were given to them by a conjugation process that allow them to acquire those abilities.”
He then makes the conclusion, that:
“ So when we do this in the lab, we're doing exactly the same thing that occurs in nature all the time. The difference is, in nature its a random process. And if an organism gets the gene from an unrelated organism and it doesn't help it in any way, doesn't confer a selective advantage, these genes are typically lost over time. If they give a selective advantage, then they're kept. When we do this on the lab, we know the advantages were conferring. It's not a random process. It's a very targeted and a very intentional process, and it allows us to manipulate the organisms. Not in wild and unpredictable ways, but in ways that we can largely predict as scientists. … In fact, all we really are doing is the same thing that nature has done for 4 billion years. ”
An article in The Economist makes the case that even humans have horizontally transferred genes in their genome:
“ Opponents of genetically modified crops often complain that moving genes between species is unnatural. Leaving aside the fact that the whole of agriculture is unnatural, this is still an odd worry. It has been known for a while that some genes move from one species to another given the chance, in a process called horizontal gene transfer. Genes for antibiotic resistance, for example, swap freely between species of bacteria. Only recently, though, has it become clear just how widespread such natural transgenics is. What was once regarded as a peculiarity of lesser organisms has now been found to be true in human beings, too. Alastair Crisp and Chiara Boschetti of Cambridge University, and their colleagues, have been investigating the matter. Their results, just published in Genome Biology, suggest human beings have at least 145 genes picked up from other species by their forebears. Admittedly, that is less than 1% of the 20,000 or so humans have in total. But it might surprise many people that they are even to a small degree part bacterium, part fungus and part alga. ”
A study from 2016 found there are even more, hundreds, of horizontally transmitted genes in the human genome.
However, another study from 2017 reanalyzed the results from the 2015 study covered by the economist, and asserted that the results were faulty.
It is well documented that horizontal gene transfer can occur from virus to human somatic cells , as in the case of human papillomavirus. However, in sexually reproducing metazoa, vertical gene transfer occurs via the more protected germline cells, so even if horizontal gene transfer occurs on soma cells, it is unlikely they will be passed on vertically. While the theory that horizontal gene transfer occurs in humans is very probable, explanations for the pathways or mechanisms for horizontal gene transfer in animals or other metazoans are mere speculation .
In the article Transfer of DNA from Bacteria to Eukaryotes, the authors describe the improbable steps needed for horizontal gene transfer to occur:
“ It is important to note that the presence of bacterial HGT signatures in eukaryotic genomes does not depend solely on the ability of bacteria to transfer DNA into the host cell. Instead, four additional major conditions should be met. First, the transferred DNA must become integrated into the host genome. Second, the foreign sequence must not be lost after rearrangements of the genome during subsequent cell divisions. Third, for multicellular eukaryotes, the transformed cell must either be fixed in the germline for genetic modification of animals and plant germlines… Finally, the integrated sequence must be preserved in the course of evolution, which is more likely to occur if the acquired gene confers a selective advantage or is at least neutral rather than deleterious. Thus, transient transformation events, which, by definition, are not retained in the genome, probably occur at a much higher rate than is suggested by the HGT signatures discovered in eukaryote genomes.”
If horizontal gene transfer “happens all the time” in nature, then perhaps Metcalf was only considering transfers that do not get passed on vertically. It appears that vertical gene transfer of horizontally transfered genes, in sexually reproducing multicellular eukaryotes may take a much longer time than the rate at which genetic engineering is occuring. Based on the articles included in the research, and input from a friend, Annie Gottlieb, a science editor, the rate at which this combination happens is unknown at this time. I will also assume that Dr. Metcalf's statement is also conversational rather than an expression of fact, since he does not provide sources to back up his claim. Perhaps one could make a rough guess based on the number of mutations that could *possibly* have originated from phages or bacterium or alga, compared to the total number of genes in the species, divided by the number of years of existence. Dr. Metcalf also claims that between 5 and 20% of genes in the human genome comes from horizontally transferred genes, and that is much higher than the “less than 1%” found in the research covered by this paper. I would be interested in reading his sources, but due to the format of the presentation, none are provided.
The World Health Organization uses relatively neutral language in making claims to assess the safety of GM foods. WHO considers how the safety assessment of GM foods is conducted, what the human health concerns are, and the possible environmental issues. Environmental concerns include:
“ …the capability of the GMO to escape and potentially introduce the engineered genes into wild populations; the persistence of the gene after the GMO has been harvested; the susceptibility of non-target organisms (e.g. insects which are not pests) to the gene product; the stability of the gene; the reduction in the spectrum of other plants including loss of biodiversity; and increased use of chemicals in agriculture. ”
There are also websites, such as http://frankenfoodfacts.blogspot.com, that cover the many positive aspects of GMO's, such as the case of a pink pineapple.
The following 2008 article by Theresa Phillips, Ph.D, titled Genetically Modified Organisms (GMOs): Transgenic Crops and Recombinant DNA Technology , covers a variety of uses of GMO's, including the case of a genetically modified salmon. A growth hormone gene was introduced into the salmon, causing a larger size:
“ A type 1 growth hormone gene injected into fertilized fish eggs results in 6.2% retention of the vector at one year of age, as well as significantly increased growth rates ”
Though a positive result, there can also be negative consequences if the genetically modified salmon were introduced in the wild:
“ …vertical gene transfer between GMOs and their wild-type counterparts have been highlighted by studying transgenic fish released into wild populations of the same species (Muir & Howard, 1999). The enhanced mating advantages of the genetically modified fish led to a reduction in the viability of their offspring. Thus, when a new transgene is introduced into a wild fish population, it propagates and may eventually threaten the viability of both the wild-type and the genetically modified organisms. ”
In the research article Feeding the world: genetically modified crops versus agricultural biodiversity , the case is made that biodiversity is a better answer than genetically modified crops:
“ The first obstacle is the claim that genetically modified crops are necessary if we are to secure food production within the next decades. This claim has no scientific support, but is rather a reflection of corporate interests. The second obstacle is the resultant shortage of research funds for agrobiodiversity solutions in comparison with funding for research in genetic modification of crops. Favoring biodiversity does not exclude any future biotechnological contributions,but favoring biotechnology threatens future biodiversity resources. An objective review of current knowledge places GM crops far down the list of potential solutions in the coming decades. We conclude that much of the research funding currently available for the development of GM crops would be much better spent in other research areas of plant science, e.g., nutrition, policy research, governance, and solutions close to local market conditions if the goal is to provide sufficient food for the world's growing population in a sustainable way. ”
Also consider, that the underlying drive for a corporation is profit rather than social good . This can also be seen in various metrics of conflicts of interest, where funding is seen to affect results of research. There are many studies covering such metrics:
Natasha Longo, a nutritionist that has consulted on public health policy in Canada, Australia, Spain, Ireland, England, and Germany, makes the statement that:
“ Although our understanding of the general biology of recombination in plants is constantly improving, we still lack the knowledge for precision engineering of plants' genes and thus GM engineering may present one of the biggest threats to human health and the environment. ”
In satisfying the requirements for this paper, I think that in order to assess the safety of GMO's, you can't just look at one article about the development of one GMO. Also, I should avoid how I “feel” about the matter, and read articles from both sides. That said, I should divulge my bias. I think genetic engineering has it's place, and I also think biodiversity has it's place. I hope both resources are used. However, I do favor locally owned small farmers, and I do think they are in competition with food manufacturing corporations, that have the potential to use GMO's as tools to favor their profits, and have the resources to lobby both governments and their citizens. The other bias I have, is that I hope to work in the research of genetic modifications. I also hope to have the funding to do this research.
The case for the safety of a genetic modification, as described by WHO, must be done on a case by case basis, and I hope through due diligence, considering the parallel concept of introducing an invasive species.
“ In the Gros Michel’s rise and fall, the banana industry struggled with the paradox that plagues all industrial agriculture crops. Natural reproduction is bad for short-term profits. The way to grow a consistent product at yields that achieve economies of scale is to stamp out the risks of diversity and imperfection that happens when genes reshuffle. To boost profit, you then grow that crop to the exclusion of less valuable species. ”
GMO's are introduced into the environment on a far larger scale than ever seen in history. In the past, cross breeding has been a small scale activity, conserving genetic diversity (https://en.wikipedia.org/wiki/Crop_diversity). Monocultured, cloned crops could have more far reaching effects, the likes of which evolution has never seen, since they are introduced suddenly over large amounts of landmass.
Genetically Engineered Crops: Experiences and Prospects
“ Genetically Engineered Crops builds on previous related Academies reports published between 1987 and 2010 by undertaking a retrospective examination of the purported positive and adverse effects of GE crops and to anticipate what emerging genetic-engineering technologies hold for the future. This report indicates where there are uncertainties about the economic, agronomic, health, safety, or other impacts of GE crops and food, and makes recommendations to fill gaps in safety assessments, increase regulatory clarity, and improve innovations in and access to GE technology. ”
This study did not recruit agronomic experts from Cuba, which practices organic and local farming . “ Due to the shortage in artificial fertilizers and pesticides, Cuba's agricultural sector largely turned organic. ”
This is a report written as a class assignment for the article below. The first paragraph is lame because it's purpose is to satisfy the assignment.
Meg Tirrell got her genome sequenced because CNBC paid for it, in exchange for her writing the article. This happened in 2015 when a whole genome sequence by Illumina cost $2900. As she described, she wasn't trying to solve any problem. To her credit, she went into it without much expectation, and, as it was more than likely, she came out of it without actually learning much. This is because so little is known about the genome at this time. Her geneticist counselor, Dr Robert Green, said at the time, that only 1 to 2 percent of people get an “actionable” result. An actionable result would be information gained requiring medical attention. Even then, there are likely to not be medical interventions that can solve whatever issue they may find.
Meg Tirrell wrote: “If it feels like genome sequencing raises more questions than it answers, that's due to a yawning gap between what sequencing technology enables us to discover, and how much we actually understand about the information we get back. ”
Only after much future research will more be understood about genomes. Her genome can then be re-evaluated as time goes on, when more is understood. Although, with current technology, the sequencing may not be perfect. She may be well to re-sequence her genome in the future. In the 10-12-17 article http://time.com/4971220/future-dna-sequencing, James Watson writes: “ The $1,000 genome is a marvelous technological feat but the current sequencing system isn’t perfect. Illumina’s scheme produces millions of tiny DNA fragments, each only about 250 letters in length, which have to be pieced together computationally to assemble the human genome jigsaw puzzle. This makes the final picture less accurate than it might be — imagine doing a puzzle with hundreds of near-identical sky-blue pieces. ”
DNA sequencing could be a private matter, even if the data is used anonymously for science. Both these points, that the data be private, and that they be available for science, can be equally important.
Consider the point made in the article titled Thanks to Genetic Testing, Everyone Could Soon Have a Pre-Existing Condition: “ Our genomes provide a window into scores of genetic risk factors that have yet to present as full-fledged pre-existing conditions. If the GOP insists that people can be charged differently depending on their current health, what’s to say they’ll stop short of asserting that we could be charged according to our genomes? ”
Labs could report all medical information to your own private data bank. The data banks could be providers, much like pharmacies. It could even be set up so that you have to allow the doctor permission to access your files on the individual labs. If the government were to invest in personal freedom, ventures like 23andme would likely participate as data banks.
There is plenty of information that scientists can use in addition to lab results and genome sequencing. China, for example, is pushing for an even more thorough accumulation of medical data: “For Wang, sequencing on its own is old hat. “Genomics is important, but it's just one piece of the puzzle,” he says. “All the complex traits. All the neurodegenerative disorders, cancer, diabetes — it's all more than genetics. If we only talk about genomics, about massive data without clinical info, that's not enough.””
Jun Wang leads the company BGI, which had 20%-30% of the world's sequencing capacity in June 2016. With the cooperation of the chinese government, that capacity is bound to increase: “Over the next five years, the government has promised to add several precision drugs and molecular-diagnosis products to the national medical-insurance list, ensuring that companies' research costs will be recouped if they lead to such a product. In the United States, biotech companies with new products can struggle to get insurance companies or the government to pay. “There is greater acceptance of sequencing and willingness to invest in it in China,” says Daly.”
For example, wired magazine wrote in the 6-26-17 article You Can Get Your Whole Genome Sequenced. But Should You? : “ For now at least, it seems like high prices and minimal insurance coverage will keep genetic testing from becoming part of the pricking and prodding routine of primary care. At a dinner to thank all the physicians who participated in the study, Vassy asked them all if they would order up genome sequencing for all their patients if they cost $1,000. Nobody raised a hand. He kept asking lower amounts. The hands stayed put. It wasn’t until he hit the $100 mark that he started to get a few takers. ”
note: skip to the discussion
Lab 11, 14 Nov 2017
Cells from an onion root tip were mounted on a slide and observed for different stages of the cell life cycle. Based on the number of cells found in different stages, an estimation was made on the amount of time a typical cell spends in each stage. Results were poor due to the difficulty of identifying cell stages among the student participants.
Cells have a life cycle that includes growth phases and mitosis. The duration of time a cell undergoes mitosis is known to be the smaller fraction of the different stages of a cell cycle. The purpose of this experiment was to estimate the duration of each step of the cell cycle, including interphase, mitosis, and that of the sub-stages of mitosis. The sub-stages of mitosis are prophase, metaphase, anaphase, and telophase. These stages can be viewed under a microscope.
During interphase, a newly formed cell undergoes growth and synthesis sub-stages. However, these stages cannot be seen because of the cell wall of the nucleus, and are thus not part of the measurements of this experiment.
During prophase (and prometaphase), the chromosomes condense into sister chromatids, the nucleolus disappears, the nuclear envelope breakes down, and the mitotic spindles start forming. The spindle apparatus starts to attach to and organizes the chromosomes, attaching some of the microtubules to the kinetochore at the centromeres.
During metaphase, the chromosomes are lined up, with each of the two kinetochores attached to a microtubule from opposite spindle poles.
During anaphase, the sister chromatids are pulled apart towards the poles. Microtubules not attached to chromosomes separate the poles and elongate the cell.
During telophase, the cell is close to dividing and cytokinesis is taking place. Two new nuclei form, and the nucleoli and the nuclear membrane begin to appear. The chromosomes decondense back into chromatin.
MATERIALS AND METHODS:
An onion root tip was sectioned and mounted on slides for viewing under the microscope. It is known that the cell cycle in the onion root tip lasts 720 minutes.
The duration of the phases of mitosis was estimated based on the percentage of cells undergoing mitosis at a particular time. A random set of 20 cells was selected, and each one added to the counts of one of the five stages covered in the introduction. Six teams participated in the experiment, each with a different sample slide.
The experiment called for a sample size of 20. This would give a confidence interval for the individual stages of mitosis at roughly 10%, based on an estimate of 5% for each of the stages of mitosis. However, the confidence interval should have been reduced by the multiple teams that took on different samples of 20 cells. With 6 teams and a total sample size of 120, the confidence interval would be 4%. 
The cell count results and time estimates for each phase of the cell cycle, in the tables below.
|Number of Cells in each phase|
|% of cells in each phase||60%||25%||5%||5%||5%||100%|
|time estimate||432||180||36||36||36||720 min|
|% of cells in each phase||23%||29%||17%||11%||21%||100%|
|time estimate||167||208||119||77||149||720 min|
Only the one Team D came close to mainstream results for onion root tip mitosis investigations.  Due to the large variation between the different teams, one of two hypothesis can be made. Hypothesis one is that the onion root tip in the lab was a mutation which spent random amounts of time in each of the phases, similar to participation by members of government congress. Hypothesis two is that the students were unable to determine the stages of each cell correctly.
Unfortunately, the confidence of the student's ability to identify the different stages was a weak point of this experiment. The slides did not greatly resemble the crystal clear example photos available in textbooks, and I think students did not have the insight to know that the amount of time a cell spends in mitosis is small compared to interphase. These contributed to students mislabeling cell stages, and adding to the wrong counts.
Future experiments could better train students to identify cell stages that do not resemble textbook quality images that are the epitome example of a particular stage. Students could be tested on stage identification before taking part in the experiment.
I would like to thank Professor Shek Sesay for his utmost patience with insubordinate students, particularly ones that use cellular enabled devices to study biology in class.