CRISPR-Cas9 History and Engineering

CRISPR-Cas9 History and Engineering A modular platform using engineered single short guide RNA to allow programming of CRISPR specificity, permitting high efficacy gene induction for analysis of gene function. The ability to control gene expression has been the key method in elucidating their respective functions, pathways, and regulatory elements; paving a way for future therapeutic applications. The two main approaches of determining gene function involve the analysis of loss-of-function (LOF) and gain-of-function (GOF) mutations. LOF involves a mutation in an allele where partial or full loss in genetic function occurs. GOF involves the introduction of a mutation which generates a new allele associated with a new function. The problem with GOF screening approaches is that theyre hindered by a requirement for large comprehensive cDNA library overexpression systems which rarely encompass the full spectrum of isoform variation. Viral expression vectors are not large enough to allow these to be cloned. LOF screening is the predominant way of analysing gene function, using techniques such as Transcription-activator-like effector nucleases (TALENs) RNA interference and Zinc finger nucleases (ZFNs). However, these are difficult to construct on a genome wide scale, unlike CRISPR-Cas9. A brief history of CRISPR-Cas9 In 1987, Ishino et al observed the presence of CRISPR repeats within bacterial genomes, but it wasnt until 2006 that Makarova proposed for its use as an adaptive immune system. Cas9 or CRISPR associated protein 9 is an endonuclease, guided by RNA and associated with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). The system functions by interrogating and cleaving foreign DNA from bacteriophages by unwinding the foreign DNA and checking its complementation to a 20 base pair spacer region on the guide RNA. If the DNA substrate is complementary to the guide RNA, cleavage of the DNA occurs (Heler R, 2015). (Jinek M, 2012) discovered that by inactivating Cas9s two catalytic domains, its DNA cleavage ability is disrupted thereby creating catalytically dead or dCas9. This provides a platform for an RNA-guided transcript activator (dCas9-activator) using a single short guided RNA (sgRNA). Engineering CRISPR-Cas9 In their article: Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex, Konermann et al develop a system using programmable DNA binding proteins for engineering synthetic transcription factors for the modulation of endogenous gene expression. This allowed GOF screening and was successful in turning on tens of thousands of individual genes in parallel. To allow rational engineering of the CRISPR-Cas9 system, the structure of the Cas9-sg-RBA-target DNA tertiary complex had to be elucidated. To do this, crystallographic studies were performed. Optimal anchoring positions were determined for the activation domains. The team settled on the addition of protein interacting RNA aptamers to the tetraloop and stemloop 2 to facilitate the recruitment of effector domains to the Cas9, as illustrated in figure 1. Fusion of the dCas9 to transcriptional activation domains converts the Cas9 nuclease into an dCas9-activator. Linking the dCas9 to domains of proteins involved in transcriptional activation and allowing CRISPR to target promotor sequences regulating transcription of particular genes provides a means of modulating natural gene expression. The efficacy of this system is low causing at most a fivefold increase in activation. Tiling the promoter region with several sgRNAs can produce a substantial transcriptional activation. Konermann et al overcame this low efficiency by turning CRISPR sgRNA into a modular platform which assembles multiple different transcriptional activators. The addition of the protein interacting RNA aptamers attracts RNA binding proteins. The complex can be used to target the transcription activation domains of different transcription factors, creating a system termed the synergistic activator mediator (SAM) by its authors. Astonishingly, this complex can induce more than 100-fold activation of genes. Parallels can be drawn with the cells natural mechanisms of gene regulation; enhancers can turn on gene expression by generating long non-coding RNAs (lncRNAs) which act as modular scaffolds, recruiting cellular machinery similarly to CRISPR. Konnermanns findings appear to mimic the lncRNAs by orchestrating the use of multiple proteins to have them work in cohesion. Current Applications The authors displayed the applications of this response by creating a library of sgRNAs, thereby allowing individual activation of over 23,000 genes. Their experiments were centred around melanoma cancer cells. PLX-4720 is a common drug treatment, capable of killing these cancerous cells. The experiment involved activation of individual genes to establish which ones would provide resistance to the killing effects of the PLX-4720 treatment. Drug resistance was determined by calculating the relative frequency of sgRNAs in melanoma cells post drug treatment. sgRNAs were correspondent to the genes involved in known drug-resistance pathways.   This verified that the SAM technique could identify biologically significant outcomes of varied gene expression. It was determined that 13 genes whose altered gene expression produced a state of drug resistance. Potential applications The significance of the findings of Konnermann et al are a new and improved programmable targeting system for DNA by which RNA sequences can be engineered to determine specificity. Through this, single sgRNA-mediated gene upregulation can be performed.This next generation of CRISPR expands the Cas9 toolbox, further engineering may take advantage of the modular nature of this system. The scaffolding allows variation in the use of aptamers, for recruitment of specific effectors It has been proposed to replace the MS2 stem loops with PP7 elements to recruit repressive elements as opposed to activators, thereby opening the possibility of bidirectional transcript control. Further research is required to determine off target effects of CRISPR and validate experiments to confirm effects of altered gene expression. This will require a detailed understanding of regulatory elements and further experiments with gene sub libraries. Future applications will involve positive and negative selectio n screens to determine genetic elements in cells.

Evil Disney, Research Response to Henry Giroux Essay

There are plenty of hotbed issues on how the Disney corporation’s sociological and socio political ideologies are embedded into their products and how they affect children, but very few ask why Disney would place hidden ideologies in their movies/shows. What reasons would Disney have to program children with outdated morals while trying desperately to uphold a model image of innocence? What practices has the disney corporation practiced that some would consider immoral or even illegal? To answer these questions the following issues must be explored in more depth: The history and actions of Disney from its inceptions to the present,Walt’s strict â€Å"moral† code along with the legacy he left behind, the policies of Disneyland along with the corporation’s political, judicial and economical power, Walt’s ties to the FBI and organized crime syndicates, and the revolving door politics in our government. Disney’s powers have allowed them unprecedented freedoms associated with a private company and the pandora’s box that is Evil Mickey. First,what is the Disney corporation and how did it come to be? It was created almost single handedly by Walter Elias â€Å"Walt† Disney. He was born on December 5, 1901, one of five children. Walt had a love for drawing cartoons early on and even made money as a child by selling some of them to family and friends. When he was 16 he joined the Red Cross as an ambulance driver for WW1 after being denied service in the army for his young age. This patriotic fervor that would later shape his cartoons. When he returned home from the war he made a few failed attempts at working in different animation companies until he and his brother, Roy, started the Disney Brothers Studio in 1923 after moving to California. The two of them prospered despite two major setbacks becoming one of the most powerful media conglomerates today. Their first setback happened In the late 1920’s. Disney learned that Winkler and her husband, Charles Mintz, had basically stolen their entire enterprise when they stole the rights to Oswald, an early character for his laugh-o-grams. Along with all but one former employee, Iwerks, who refused to leave Disney, hopefully winning him employee of the year. (biography. com) Charles was unsatisfied with the production costs for earlier creations. When Oswald became highly successful, Mintz hired all of the former employees thereby giving the rights to the later formed Universal picture’s first creation. According to his biography, during this time Walt’s wife was pregnant with their first child and Walt went into a deep depression claiming it on â€Å"financial stress†. (sito, ch5) There is a lot of speculation about this time in Walt’s life. Some say that the Mishpucka and the organized mob had an influence into this(babbit); the mob, who at the time were powerful in the freezone of Hollywood, controlling unions, local law, real estate, film sets, etc. Time Magazine, Nov. 1, 1943, wrote, â€Å"In the witness chair in Manhattan’s Federal Court sat bland, Wily Willie Bioft (pronounced Buy-off), blackmailer, panderer, labor leader, and now star witness against eight ex-pals, who are charged with shaking down $1 million from the movie industry†¦ Question: Was it true that Bioft once had a five-year plan for taking over 20% of Hollywood’s profits-and eventually 50% interest In the studios themselves? Bioff (wistfully): â€Å"If we’d lasted that long, we would have. Question: â€Å"Did you ever say you were boss of Hollywood and could make producers do whatever you wanted? † Bioff: â€Å"Yes-and I could make them dance to my tune. † Although Bioff rolled over on his pals and ended up getting car bombed later there is no proof that Walt ever worked with them or against them. The only link between Walt and the Mishpucka/Mafia is Mafia associate, Henry Cohn. After the first setback with Mintz Walt was heavily in debt and needed some way to bounce back his business after losing nearly everything but his mind. Henry offered to give Walt a loan. The thought of the mafia looming over his head is one of the reasons stated for his severe depression (Rasmussen&Sito) The theft of Oswald, was a blessing in disguise that lead to the creation of Mickey Mouse and later to create the worlds first full length animated movie in 1937, Snow White and the Seven Dwarfs, which won 8 Oscars and made 1. 5 million despite being in the midst of an economic meltdown. (biography. com) Opening up Disney to a whole new realm of marketing and showing Walt the popularity and power of his studios. The second setback was the strike of 1941. Which was instigated by the firing of former head of the Federation of Screen Cartoonists and later leader of the Screen Cartoonists Guild, Art Babbitt on May 28, which caused 200 animators to go on strike almost immediately. (Sito&babbit) Walt early on had discovered that propaganda and public image were key to selling media, which is why he was so studious about keeping the interior of the workplace â€Å"Christian†. Walt had created the Federation of Screen artists to better control his workers, which was a union that regularly met with him on issues such as wage increases, better hours, less footage quotas, and better work environments. Since Walt was notorious for being a bit cruel and demanding in the office. (rasmussen) Though as soon as Art learned this he created the Screen Cartoonists Guild whose recruiting patterns were not the nicest methods possible but it did get them all to join â€Å"There were these tough union guys who said we couldn’t enter the door unless we joined, so we did. Under protest I joined. Because of the new job classification my salary doubled overnight. So i can’t complain about the union. † (sito) This was a time when union laws were strong under Teddy Roosevelt, more often than not the law would favor the people over the corporation. This was also a time when many Mob bosses were often also Union bosses so it was a give and take situation. Unions were now viewed as less as a helpful barrier between workers and the corporations to something evil, something, Communist. With the Red scare well under way and people were mixing socialist ideologies with communism. Though at this time the idea of communism was quite popular the way patsies were during the prohibition age. It was doing quite well in the underground of society. Even within Disney there was communism, Walt even thought communist agitators were the culprit to the 1941 strike. sito) This all of course is the end result of years of oppressive rule under Walt along with strengthening unions demanding better pay and working conditions. Walt hated those whose ideas drifted more towards the left wing which would fit the image of the highly conservative man he was. In the office an employee could be fired on the spot for any kind of social infraction. If an employee cursed or made any sort of rude engagements with a woman they would be forced to leave and of course there was no drinking allowed in his office. Disney employees under Walt had to get a hall pass to go to the restroom or get up at all† Of course walt was known for his rages, often times screaming at artists who had not met his quotas of 30 to sometimes 60 feet of film in one day. He was also known to be a heavy drinker in his officeâ€Å"You could smell the gin on his breath everyday after lunch† (Sito) Walt testified about the strike in the hearings of the House Committee on Un-American Activities about this and much more calling them â€Å"Communist agitators†, among other less decent things. sito) This of course started the well documented relationship between Walt and the FBI from 1940 till his death. He was promoted to full Special Agent in Charge Contact, which meant he had people under him finding communists and saboteurs, Ronald Reagan being one of his fellow operatives under T-10. He and Reagan Blacklisted many writers, actors and artists for having a differing political belief as that of the reigning government. The FBI also gained access to Disneyland to use as a platform for whatever they want. There is talk online on blogs that the FBI still use Disneyland to monitor foreign nationals entering the park since the threat of communism no longer applies. (Herbert&Sito&Babbit&Rasmussen) The relationship Walt had with the FBI and the ability to blacklist anybody he wanted may subscribe to Disney’s amazing rise during this time as a media powerhouse(but thats only speculation) This cozy relationship between Disney and the government has continued through the wonderful world of the revolving door politics. A sad modern relationship that is so commonly seen between politics and private companies. Many who are in executives in Disney will enter into politics and gain high levels positions starting as lobbyists and squeezing their way into actual positions of power in the government. Marsha Macbride for example, worked as an FCC chief of staff to chairman powell for years before sometime in the late 1990’s she started to lobby for Disney and was a major advisor in the clinton administration as well as during the merger of time warner and AOL, which Disney ended up winning and gaining rights along with cash settlements in 2003. The next year she is working as the Executive Vice President for Legal and Regulatory Affairs at the National Association of Broadcasters and still is. (gov relations 1-3) Another is Lisa Caputo who served citigroup, disney vp of communications and as a bill clinton staffer (gov relations 5)or Susan Fox who worked as an fcc advisor before going to work for disney as an advisor between the government on their divisions such as the Disney Interactive Media Group, Disney Channel, the Disney ABC Cable Networks, ABC and ESPN. (gov relations 4) Others include George mitchell who worked in on Board of Directors as well as a US senator. Eileen O’connor was a news producer for ABC but is now working in a US embassy. Dennis Hightower a former Sr. executive officer transferred to Dep Secretary of commerce. (gov relations 3&5)There is even a department of Disney called â€Å"Disney government relations† that has a direct link to another department within the FCC. After calling a few times and getting different information I got this â€Å"we work with our partners in government such as the AFA or the FCC to get better standards of broadcasting for the Disney corporation. Basically legally bribing politicians to lower their standards and to take Disney’s side in lawsuits and corporate takeovers. Now a days after Walt’s death Disney has expanded to epic proportions in the media industry. The public face of cuddly animals, children’s fairy tales and building theme parks â€Å"It also owns six motion picture studios, ABC television network and its 226 affiliated stations, multiple cable television networks, 227 radio stations, four music companies, three cruise lines, theatrical production companies, publishing houses, multiple educational shorts, 15 magazine titles and five video game development studios. (gabler)It became the cultural pervader of a perfect world with everything that it put its name on. But there is a darker side to Disney that is up for debate. The side of Disney that is bigoted, American exceptionalist, sexist, racist, and a purveyor of pain and suffering. The propaganda machine that is Disney today. Propaganda has always been an important part in any government, from mummers playing the latest military conquest to Disney’s subliminal messages of obedience in children’s movies. Producers construct fantasy societies that seem to run in perfect harmony where each worker and is happy to work their menial jobs and never aspire to try to raise themselves above their born level. This is seen in the workers of Antz, Sleeping Beauty and Oz. Always with an all powerful â€Å"just ruler†. These stories lead children into blindly following their leaders down the yellow brick road to the great and powerful Oz. â€Å"Workers are quite happy to â€Å"serve the rich and privileged, never questioning their subordinate position. (giroux pg 102). Another example is the Chicken little movie in 1938, where the people are the chickens, the farmer the government and the fox being intellectuals (with his psychology book) Programming americans, along with all of the other(now banned) wartime disney movies that seep out Walts own personal bigoted morals. Another example is the â€Å"Thrifty Pigs† which is trying to get people to buy war bonds to stop â€Å"the big bad wolf†, or â€Å"Home Defense† which makes fun of the French soldiers as being incompetent. All of these and countless others that were formed to get the people to fight in a war that we had no part in until Pearl Harbor. Or the worst one, â€Å"The spirit of the 43† â€Å"taxes to fight the axis† â€Å"Dont spend your money on things you dont need, give it to the government. † Nothing like big brother Disney. But this side of Disney stops, at least blatantly, after Walts death in 1966 when it became a modern heartless bureaucratic corporation. Yet hidden messages are still within modern movies, though with less obvious programming. â€Å"vciolinguistic construction† of social dominance and inferiority in which characters who use mainstream American English tend to be associated with â€Å"strongly positive actions and motivations†Ã¢â‚¬  [pg 102 of Giroux] While the antagonist is often of another nationality such as the British voiced Scar or any voice that isn’t middle class Suburbia speech, as seen in Ursula’s louisiana sw amp accent. This pushes children to view those of the lower social class, dark skinned people or foreigners to be â€Å"evil† and become xenophobic and racist towards anything different than themselves. Disney goes a step further and makes a culture of consumerism, â€Å"’without tenements or poverty or urban class conflict†¦ It’s a native white Protestant dream of a world without blacks or immigrants. â€Å", â€Å"image of small towns characterized by cheerful commerce, with barbershop quartets and ice cream sundaes and glorious parades. â€Å"(giroux pg 96) Everything media wise that Disney produces ends up becoming a cog in the Disney corporate machine with imaginers spewing out product after product to sell to children and increase profits. Children see a movie and want to emulate the role models perceived, then a commercial comes on after Mickey mouse club house for the newest enchanted Princess gown or Woody’s costume and the kids ask their parents to get it. Children have not just copied the looks of the protagonists in Disney stories but have come to expect those fantasies to play out in real life, when the real world isn’t such a happy place and has no room for such fantasy. People end up growing up focusing on material items and their personal lives instead of focusing on the fixable issues, calling it the American dream. This ideology of apathy is a big problem now with revolving door politics, politicians who promise the same things over and over and never fix them and blame the other side, failing infrastructure, falling public school standards and debt through the roof. Not saying that its all Disney’s fault, of course not. Though almost every American child has seen at least one disney movie, it’s almost impossible not to be sucked into Disney’s sway since they even show them often in public schools as educational videos never delving deeper. As a social manipulator Disney has taken the fight out of us. Programing us to follow American exceptionalism since the American was the hero is portrayed morally just whilst his/her enemy spoke in an exotic accent along with generating xenophobism of other cultures. Reinforcing children to follow the status quo and not to learn about the rest of the worlds culture outside their own narrow sphere of friends and family, enclosing ourselves in a media shell while proliferating American’s culture and moral values to other nations, who view disney as the true america. Disneyland †¦ it is the â€Å"real† country, all of â€Å"real† America† -Jean Baudril ­lard [pg 95 of giroux. ] To be fair, countless other â€Å"children’s shows† show â€Å"non moral/family friendly broadcasting, especially now. Watching Spongebob as a child or the modern shows such as Adventure Time or the Regular Show and then seeing them as an adult. Explaining why my mother would laugh at certain parts of the shows that I didn’t laugh at. I notice now the abundance of sexual jokes I missed as a kid and the references to drugs, crimes, war, and all the other stuff thats messed up with the world. So this kind of hidden messages has been deeply imbedded into all sorts media, they are adults working on childrens shows after all, so i doubt they’d be â€Å"mature†. What is evil about Disney is the corporation and those leading it, not the movies that they put their name on. This culture of consumerism that the corporation has exacerbated has evolved into a new network with companies working together to sell a common goal.

Time Management Skills - Free Essay Example

Sample details Pages: 3 Words: 801 Downloads: 10 Date added: 2019/05/29 Category Management Essay Level High school Tags: Time Management Essay Did you like this example? What are you doing right now? What do you wish you could be doing right now? What other things could you be doing right now? These are all good questions that all come to one common term. Time management. We all know the feeling of maybe I should do this before I do that. Don’t waste time! Our writers will create an original "Time Management Skills" essay for you Create order All people can agree efficiency is what makes the world spin but the ideals can be scaled at miniscule or gargantuan levels in the sense you can be efficient with doing the dishes or a multi billion dollar company can be efficient with the production of those plates. Jeff bezos is a multi billionaire and the owner of Amazon an almost trillion dollar company but he does this because he is very good at predicting things and somewhat guessing. He only owns 16% when it comes to the company itself but he is using his knowledge of management and putting all of his time towards the most important aspect of his company. Time management skills can be used to become more efficient and less stressful in the long run. The ability to be able to get things done on a schedule makes life go by faster meaning one can use more time to worry less about smaller inconveniences. Forty fifth president of the United States of America, Donald John Trump Created The Art of The Deal and in that book he talked about the ways he used his time in such a way that resulted in productivity in his life. Like what trump mentioned in his book pertaining to this topic was, the time you spend doing something doesnt matter, itrs what comes from that time spent. He also mentioned how his jobs had significantly lower prices (cost for them) than other companies due to them being ahead of schedule. Using those tools to better your time and to get ahead of other things that you might be focused on. These all are great ways to help someone out with their most important value and to get more enjoyment out of what they put in to something. Clincher: What is the importance of this subtopic? The organisation of your time is an efficient way to make your life more productive and useful due to what you know and when something is happening. Stress can overwhelm anything due to over thinking and over/ under production and meeting things at certain dates so itrs not bad to just think. Matt Mayberry is a motivational speaker and a CEO of a company, and is very successful at both, but in one of his articles about Time management he wrote about how someone can Take a walk, go to the gym, get some fresh air or take that sick day you have been holding out on (Mayberry). This quote is very good for someone who over works and never has time to themselves. People have needed some time to think and doing some of the suggested things Matt said is a good way to clear your head so you can get back on track. By doing this people get to working harder on one thing because the negatives have been cleared from their head. Mayberryrs article is very helpful in listing ways you can use time correctly or in your favor but a great listen would be Time management Is Key To Getting Work Done. This is a great podcast between two great women in Radio/Podcasts Renee Montagne and Lucy Kellaway are great examples of everyday people. In this podcast these ladies talk about their daily struggles. Lucy mentioned that her addiction to reading emails gets in the way of her other important tasks due to how stressful they can be. This is a great point because small things like emails or texts can suck us in to doing something for a long period of time without being productive. People tend to get off topic and lose productivity due to these small obstacles and stress due to them. This whole process is a waste of time and unnecessary . The effective use of time management skills are what help people and businesses run faster and with less problems. Most people Have smaller obstacles that when piled up can become major and hinder their full potential and need way to get around those things. Schedules can really help as well due to having everything in a position instead of just saying whatrs next people already have a game plan. Most people can agree that if you use any management skills not just time management skills your life will be a whole lot smoother. People as a whole are better impacted when not only people work together but have specialized ways they can work themselves with those other people to reach the end goal.

Children s Academic And Social Competencies - 1549 Words

When every child has the chance to meet his or her full potential, families, communities, and the economic future of the United States is reinforced. Surprisingly, one in four children in the United States living in low-income households enters kindergarten not equipped to learn and, as a result, fall behind from the start. â€Å"Children s academic and social competencies at kindergarten entry are important predictors of success throughout school; children who enter school not ready to learn struggle with academic difficulties and [can] manifest social and behavior problems in later school years† (Duncan et al., 2007; Ladd, 2006). â€Å"When a young child enters kindergarten ready for school, there is an 82 percent chance that child will†¦show more content†¦Ã¢â‚¬Å"Children with early developmental delays are at heightened risk for behavior problems and co-morbid psychopathology [; relating to more than one disorder or disease occurring at the same time]† (G erstein, E. D.et al, 2011). America greatly under-invest in early childhood education and, like so many political battles these days, the disparity between bipartisan support for preschool education programs and cutting thousands of kids from programs, such as, Head Start, Early Head Start and the Child Care and Development Block Grant programs (CCDBG) defies reason and common sense. There are those within our current political leadership that feel it should be left up to each individual state, as to how they want to distribute educational subsidies, leaving lower socioeconomic communities concerned with budget cuts and where they will be getting their funding from to educate their children; sending these neighborhoods in panic to find financial support elsewhere and without delay. These programs and other programs similar are in jeopardy of not receiving adequate backing as it will be left to each state to decide the importance of early childhood education and early intervention . 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