From The University of Washington (US) School of Medicine: “AI-designed protein awakens silenced genes one by one”

From The University of Washington (US) School of Medicine

March 1, 2022 “For immediate release” [but only released today 3.14.22]

Leila Gray
206.475.9809
leilag@uw.edu

Technique allows researchers to toggle on individual genes that regulate cell growth development and function.

Hanelle Ruohola-Baker discusses regulation of gene activity research with Shiri Levy in her lab. Credit: Thatcher Heldring/ISCRM.

By combining CRISPR technology with a protein designed with artificial intelligence, it is possible to awaken individual dormant genes by disabling the chemical “off switches” that silence them. Researchers from the University of Washington School of Medicine in Seattle describe this finding in the journal Cell Reports.

The approach will allow researchers to understand the role individual genes play in normal cell growth and development, in aging, and in such diseases as cancer, said Shiri Levy, a postdoctoral fellow in UW Institute for Stem Cell and Regenerative Medicine (ISCRM) and the lead author of the paper.

“The beauty of this approach is we can safely upregulate specific genes to affect cell activity without permanently changing the genome and cause unintended mistakes,” Levy said.

The project was led by Hannele Ruohola-Baker, professor of biochemistry and associate director of ISCRM. The AI-designed protein was developed at the UW Medicine Institute for Protein Design (IPD) under the leadership of David Baker, also a professor of biochemistry and head of The University of Washington Institute for Protein Design.

U Washington,Dr. David Baker, Baker Lab.

The new technique controls gene activity without altering the DNA sequence of the genome by targeting chemical modifications that help package genes in our chromosomes and regulate their activity. Because these modifications occur not in, but on top of genes, they are called epigenetic, from the Greek epi “over” or “above” the genes. The chemical modifications that regulate gene activity are called epigenetic markers.

Scientists are particularly interested in epigenetic modifications because not only do they affect gene activity in normal cell function, epigenetic markers accumulate with time, contribute to aging, and can affect of the health of future generations as we can pass them on to our children.

In their work, Levy and her colleagues focused on a complex of proteins called PRC2 that silences genes by attaching a small molecule, called a methyl group, to a protein that packages genes called histones. These methyl groups must be refreshed so if PRC2 is blocked the genes it has silenced. it can be reawakened.

PRC2 is active throughout development but plays a particularly important role during the first days of life when embryonic cells differentiate into the various cell types that will form the tissues and organs of the growing embryo. PRC2 can be blocked with chemicals, but they are imprecise, affecting PRC2 function throughout the genome. The goal of the UW researchers was to find a way to block PRC2 so that only one gene at a time would be affected.

To do this, David Baker and his colleagues use AI to create a protein that would bind to PRC2 and block a protein the PRC2 uses to modify the histones. Ruohola-Baker and Levy then fused this designed protein with a disabled version of a protein called Cas9.

Cas9 is the protein used in the gene editing process called CRISPR. Cas9 binds and uses RNA as an address-tag. The system allows scientists, by synthesizing a specific “address-tag” RNA, to bring Cas9 to a precise location in genome and therefore cut and splice genes at specific sites. In this experiment, however, the cutting function of the Cas9 protein is disabled so the genomic DNA sequence is unaltered. As a result, it’s called dCas9, for “dead.” However, the Cas9 function as a “vehicle” to deliver cargo to a specific location remains active. The AI-designed blocking protein was the cargo of the dCas9-RNA construct. “dCas9 is like UBER,” says Levy, “It will take you anywhere on the genome you want to go. The guide RNA is like a passenger, telling the UBER where to go.”

In the new paper, Levy and her colleagues show that by using this technique they were able to block PRC2 and selectively turn on four different genes. They were also able to show they could transdifferentiate induced pluripotent stem cells to placental progenitor cells by simply turning on two genes.

“This technique allows us to avoid bombarding cells with various growth factors and gene activators and repressors to get them to differentiate,” Levy said. “Instead, we can target specific sites on the gene transcription promoters’ region, lift those marks and let the cell do the rest in an organic, holistic manner.”

Finally, the researchers were able to show how the technique can be used to find the location of specific PRC2-controlled regulatory regions from where individual genes are activated. The location of many of these are unknown. In this case, they identified a promoter region—called a TATA box—for a gene called TBX18. Although current thinking is that these promotor regions are close to the gene, within in 30 DNA base pairs, they found for this gene the promoter region was more than 500 base pairs away.

“This was a very important finding,” said Ruohola-Baker. “TATA boxes are scattered throughout the genome, and current thinking in biology is that the important TATA boxes are very close to the gene transcription site and the others don’t seem to matter. The power of this tool is that it can find the critical PRC2 dependent elements, in this case TATA boxes that matter.”

Epigenetic modifications decorate broad regions of the genome in normal and abnormal cells. However, the minimal functional unit for the epigenetic modification remains poorly understood, Ruohola-Baker notes, “With these two advances, AI-designed proteins and CRISPR technology, we can now find the precise epigenetic marks that are important for gene expression, learn the rules and utilize them to control cell function, drive cell differentiation and develop 21st century therapies.”

This work was supported by grants from the National Institutes of Health (R01GM097372, R01GM97372-03S1, R01GM083867, 1P01GM081619, R42HG010855, U01CA246503), Department of Defense (PR203328 W81XWH-21-1-0006), American Heart Association (19IPLOI34760143) and the Washington Research Foundation, ISCRM Fellows program, and Brotman Baty Institute (BBI) for Precision Medicine.

See the full article here .

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The University of Washington School of Medicine (UWSOM) is a large public medical school in the northwest United States, located in Seattle and affiliated with the University of Washington. According to U.S. News & World Report’s 2022 Best Graduate School rankings, University of Washington School of Medicine ranked #1 in the nation for primary care education, and #7 for research.

UWSOM is the first public medical school in the states of Washington, Wyoming, Alaska, Montana, and Idaho. The school maintains a network of teaching facilities in more than 100 towns and cities across the five-state region. As part of this “WWAMI” partnership, medical students from Wyoming, Alaska, Montana, and Idaho spend their first year and a half at The University of Wyoming (US), The University of Alaska-Anchorage (US), Montana State University (US), or The University of Idaho (US), respectively. In addition, sixty first-year students and forty second-year students from Washington are based at Gonzaga University (US) in Spokane. Preference is given to residents of the WWAMI states.

The University of Washington (US) is one of the world’s preeminent public universities. Our impact on individuals, on our region, and on the world is profound — whether we are launching young people into a boundless future or confronting the grand challenges of our time through undaunted research and scholarship. Ranked number 10 in the world in Shanghai Jiao Tong University rankings and educating more than 54,000 students annually, our students and faculty work together to turn ideas into impact and in the process transform lives and our world. For more about our impact on the world, every day.

So what defines us —the students, faculty and community members at the University of Washington? Above all, it’s our belief in possibility and our unshakable optimism. It’s a connection to others, both near and far. It’s a hunger that pushes us to tackle challenges and pursue progress. It’s the conviction that together we can create a world of good. Join us on the journey.

The University of Washington (US) is a public research university in Seattle, Washington, United States. Founded in 1861, University of Washington is one of the oldest universities on the West Coast; it was established in downtown Seattle approximately a decade after the city’s founding to aid its economic development. Today, the university’s 703-acre main Seattle campus is in the University District above the Montlake Cut, within the urban Puget Sound region of the Pacific Northwest. The university has additional campuses in Tacoma and Bothell. Overall, University of Washington encompasses over 500 buildings and over 20 million gross square footage of space, including one of the largest library systems in the world with more than 26 university libraries, as well as the UW Tower, lecture halls, art centers, museums, laboratories, stadiums, and conference centers. The university offers bachelor’s, master’s, and doctoral degrees through 140 departments in various colleges and schools, sees a total student enrollment of roughly 46,000 annually, and functions on a quarter system.

University of Washington is a member of the Association of American Universities(US) and is classified among “R1: Doctoral Universities – Very high research activity”. According to the National Science Foundation(US), UW spent $1.41 billion on research and development in 2018, ranking it 5th in the nation. As the flagship institution of the six public universities in Washington state, it is known for its medical, engineering and scientific research as well as its highly competitive computer science and engineering programs. Additionally, University of Washington continues to benefit from its deep historic ties and major collaborations with numerous technology giants in the region, such as Amazon, Boeing, Nintendo, and particularly Microsoft. Paul G. Allen, Bill Gates and others spent significant time at Washington computer labs for a startup venture before founding Microsoft and other ventures. The University of Washington’s 22 varsity sports teams are also highly competitive, competing as the Huskies in the Pac-12 Conference of the NCAA Division I, representing the United States at the Olympic Games, and other major competitions.

The university has been affiliated with many notable alumni and faculty, including 21 Nobel Prize laureates and numerous Pulitzer Prize winners, Fulbright Scholars, Rhodes Scholars and Marshall Scholars.

In 1854, territorial governor Isaac Stevens recommended the establishment of a university in the Washington Territory. Prominent Seattle-area residents, including Methodist preacher Daniel Bagley, saw this as a chance to add to the city’s potential and prestige. Bagley learned of a law that allowed United States territories to sell land to raise money in support of public schools. At the time, Arthur A. Denny, one of the founders of Seattle and a member of the territorial legislature, aimed to increase the city’s importance by moving the territory’s capital from Olympia to Seattle. However, Bagley eventually convinced Denny that the establishment of a university would assist more in the development of Seattle’s economy. Two universities were initially chartered, but later the decision was repealed in favor of a single university in Lewis County provided that locally donated land was available. When no site emerged, Denny successfully petitioned the legislature to reconsider Seattle as a location in 1858.

In 1861, scouting began for an appropriate 10 acres (4 ha) site in Seattle to serve as a new university campus. Arthur and Mary Denny donated eight acres, while fellow pioneers Edward Lander, and Charlie and Mary Terry, donated two acres on Denny’s Knoll in downtown Seattle. More specifically, this tract was bounded by 4th Avenue to the west, 6th Avenue to the east, Union Street to the north, and Seneca Streets to the south.

John Pike, for whom Pike Street is named, was the university’s architect and builder. It was opened on November 4, 1861, as the Territorial University of Washington. The legislature passed articles incorporating the University, and establishing its Board of Regents in 1862. The school initially struggled, closing three times: in 1863 for low enrollment, and again in 1867 and 1876 due to funds shortage. University of Washington awarded its first graduate Clara Antoinette McCarty Wilt in 1876, with a bachelor’s degree in science.

19th century relocation

By the time Washington state entered the Union in 1889, both Seattle and the University had grown substantially. University of Washington’s total undergraduate enrollment increased from 30 to nearly 300 students, and the campus’s relative isolation in downtown Seattle faced encroaching development. A special legislative committee, headed by University of Washington graduate Edmond Meany, was created to find a new campus to better serve the growing student population and faculty. The committee eventually selected a site on the northeast of downtown Seattle called Union Bay, which was the land of the Duwamish, and the legislature appropriated funds for its purchase and construction. In 1895, the University relocated to the new campus by moving into the newly built Denny Hall. The University Regents tried and failed to sell the old campus, eventually settling with leasing the area. This would later become one of the University’s most valuable pieces of real estate in modern-day Seattle, generating millions in annual revenue with what is now called the Metropolitan Tract. The original Territorial University building was torn down in 1908, and its former site now houses the Fairmont Olympic Hotel.

The sole-surviving remnants of Washington’s first building are four 24-foot (7.3 m), white, hand-fluted cedar, Ionic columns. They were salvaged by Edmond S. Meany, one of the University’s first graduates and former head of its history department. Meany and his colleague, Dean Herbert T. Condon, dubbed the columns as “Loyalty,” “Industry,” “Faith”, and “Efficiency”, or “LIFE.” The columns now stand in the Sylvan Grove Theater.

20th century expansion

Organizers of the 1909 Alaska-Yukon-Pacific Exposition eyed the still largely undeveloped campus as a prime setting for their world’s fair. They came to an agreement with Washington’s Board of Regents that allowed them to use the campus grounds for the exposition, surrounding today’s Drumheller Fountain facing towards Mount Rainier. In exchange, organizers agreed Washington would take over the campus and its development after the fair’s conclusion. This arrangement led to a detailed site plan and several new buildings, prepared in part by John Charles Olmsted. The plan was later incorporated into the overall University of Washington campus master plan, permanently affecting the campus layout.

Both World Wars brought the military to campus, with certain facilities temporarily lent to the federal government. In spite of this, subsequent post-war periods were times of dramatic growth for the University. The period between the wars saw a significant expansion of the upper campus. Construction of the Liberal Arts Quadrangle, known to students as “The Quad,” began in 1916 and continued to 1939. The University’s architectural centerpiece, Suzzallo Library, was built in 1926 and expanded in 1935.

After World War II, further growth came with the G.I. Bill. Among the most important developments of this period was the opening of the School of Medicine in 1946, which is now consistently ranked as the top medical school in the United States. It would eventually lead to the University of Washington Medical Center, ranked by U.S. News and World Report as one of the top ten hospitals in the nation.

In 1942, all persons of Japanese ancestry in the Seattle area were forced into inland internment camps as part of Executive Order 9066 following the attack on Pearl Harbor. During this difficult time, university president Lee Paul Sieg took an active and sympathetic leadership role in advocating for and facilitating the transfer of Japanese American students to universities and colleges away from the Pacific Coast to help them avoid the mass incarceration. Nevertheless many Japanese American students and “soon-to-be” graduates were unable to transfer successfully in the short time window or receive diplomas before being incarcerated. It was only many years later that they would be recognized for their accomplishments during the University of Washington’s Long Journey Home ceremonial event that was held in May 2008.

From 1958 to 1973, the University of Washington saw a tremendous growth in student enrollment, its faculties and operating budget, and also its prestige under the leadership of Charles Odegaard. University of Washington student enrollment had more than doubled to 34,000 as the baby boom generation came of age. However, this era was also marked by high levels of student activism, as was the case at many American universities. Much of the unrest focused around civil rights and opposition to the Vietnam War. In response to anti-Vietnam War protests by the late 1960s, the University Safety and Security Division became the University of Washington Police Department.

Odegaard instituted a vision of building a “community of scholars”, convincing the Washington State legislatures to increase investment in the University. Washington senators, such as Henry M. Jackson and Warren G. Magnuson, also used their political clout to gather research funds for the University of Washington. The results included an increase in the operating budget from $37 million in 1958 to over $400 million in 1973, solidifying University of Washington as a top recipient of federal research funds in the United States. The establishment of technology giants such as Microsoft, Boeing and Amazon in the local area also proved to be highly influential in the University of Washington’s fortunes, not only improving graduate prospects but also helping to attract millions of dollars in university and research funding through its distinguished faculty and extensive alumni network.

21st century

In 1990, the University of Washington opened its additional campuses in Bothell and Tacoma. Although originally intended for students who have already completed two years of higher education, both schools have since become four-year universities with the authority to grant degrees. The first freshman classes at these campuses started in fall 2006. Today both Bothell and Tacoma also offer a selection of master’s degree programs.

In 2012, the University began exploring plans and governmental approval to expand the main Seattle campus, including significant increases in student housing, teaching facilities for the growing student body and faculty, as well as expanded public transit options. The University of Washington light rail station was completed in March 2015, connecting Seattle’s Capitol Hill neighborhood to the University of Washington Husky Stadium within five minutes of rail travel time. It offers a previously unavailable option of transportation into and out of the campus, designed specifically to reduce dependence on private vehicles, bicycles and local King County buses.

University of Washington has been listed as a “Public Ivy” in Greene’s Guides since 2001, and is an elected member of the American Association of Universities. Among the faculty by 2012, there have been 151 members of American Association for the Advancement of Science, 68 members of the National Academy of Sciences(US), 67 members of the American Academy of Arts and Sciences, 53 members of the National Academy of Medicine(US), 29 winners of the Presidential Early Career Award for Scientists and Engineers, 21 members of the National Academy of Engineering(US), 15 Howard Hughes Medical Institute Investigators, 15 MacArthur Fellows, 9 winners of the Gairdner Foundation International Award, 5 winners of the National Medal of Science, 7 Nobel Prize laureates, 5 winners of Albert Lasker Award for Clinical Medical Research, 4 members of the American Philosophical Society, 2 winners of the National Book Award, 2 winners of the National Medal of Arts, 2 Pulitzer Prize winners, 1 winner of the Fields Medal, and 1 member of the National Academy of Public Administration. Among UW students by 2012, there were 136 Fulbright Scholars, 35 Rhodes Scholars, 7 Marshall Scholars and 4 Gates Cambridge Scholars. UW is recognized as a top producer of Fulbright Scholars, ranking 2nd in the US in 2017.

The Academic Ranking of World Universities (ARWU) has consistently ranked University of Washington as one of the top 20 universities worldwide every year since its first release. In 2019, University of Washington ranked 14th worldwide out of 500 by the ARWU, 26th worldwide out of 981 in the Times Higher Education World University Rankings, and 28th worldwide out of 101 in the Times World Reputation Rankings. Meanwhile, QS World University Rankings ranked it 68th worldwide, out of over 900.

U.S. News & World Report ranked University of Washington 8th out of nearly 1,500 universities worldwide for 2021, with University of Washington’s undergraduate program tied for 58th among 389 national universities in the U.S. and tied for 19th among 209 public universities.

In 2019, it ranked 10th among the universities around the world by SCImago Institutions Rankings. In 2017, the Leiden Ranking, which focuses on science and the impact of scientific publications among the world’s 500 major universities, ranked University of Washington 12th globally and 5th in the U.S.

In 2019, Kiplinger Magazine’s review of “top college values” named University of Washington 5th for in-state students and 10th for out-of-state students among U.S. public colleges, and 84th overall out of 500 schools. In the Washington Monthly National University Rankings University of Washington was ranked 15th domestically in 2018, based on its contribution to the public good as measured by social mobility, research, and promoting public service.

From Center For Humans & Nature: “What the Planet of the Apes Franchise Teaches Us About Evolution”

Center For Humans & Nature

9.20.17
William B. Miller, Jr. M.D.

The Planet of the Apes series is one of the most successful franchises in Hollywood history. Since 1968, and over the course of six attention-grabbing movies, nearly 2 billion dollars has flowed from audiences to Hollywood.

War for the Planet of the Apes is a 2017 American science fiction film directed by Matt Reeves (Dawn of the Planet of the Apes; Let Me In; Cloverfield) from a screenplay co-written with Mark Bomback (Total Recall; The Night Caller).

In the most recent films of that series, the narrative begins with ALZ-12, a drug designed to cure Alzheimer’s. In the movie, that drug was based on a specific type of virus, known as a retrovirus. Retroviruses easily infect cells but can also make a special copy of their own DNA that can be inserted into a genome, which is our basic central system of heredity. In the case of the apes that were exposed to the drug, the retrovirus used in the drug successfully inserts into their DNA and they are permanently and dramatically changed.

Sounds like a great science fiction plot, right? Not entirely. This type of retroviral infection has happened throughout evolutionary history. It’s even happening right now. For example, HIV is a retrovirus. Another example, is Koala retrovirus, which is very similar to HIV. An important difference, especially if you are a Koala, is that this particular retrovirus has successfully inserted itself into the Koala genome and is now a part of their heredity DNA. This is a particular surprise since it has only been a very few years that we have been aware that this type of infectious insertion could happen and an instance of it has already been documented in real-time.

Over the course of evolution, we, as humans, have not been spared. There is substantial evidence of overwhelming viral contributions to our human genome. It has been estimated that as much as 50% our genome can be considered viral in origin with at least 9% of it known to be specifically retroviral in origin.

In the most recent movie in the franchise, War for the Planet of the Apes, a worldwide retroviral infection proves a boon to the apes. Non-human primates, and particularly the apes, became smarter and stronger. They gain the ability to speak. Their reflexes and endurance are improved. Even their eye color is changed. The outcome for the humans? Not so good. That same virus caused a mass human extinction. The few remaining humans that survive are immune but, arguably, not as clever as the apes.

Of course, nothing like this has actually happened right before our eyes. But the mechanism by which these evolutionary processes are portrayed is not scientifically unreasonable if you are willing to accept the growing scientific evidence that the standard Darwinian narrative of evolution needs some contemporary adjustment.

Certainly, the Darwinian interpretation of evolution remains preeminent. Darwinists insist that evolution proceeds by tiny changes through random genetic variations. Once those genetic accidents occur, the direction of evolution is shaped by natural selection. If the changes promote an organism that is more ‘fit’, meaning that it can reproduce more successfully than another, then this random mutation and the change it allows can continue. Crucially though, for Darwinists, evolutionary changes are necessarily small in scale.

Yet, there are a growing number of scientists that think otherwise. They believe that evolution can move in jumps from time to time. And pertinent to War for the Planet of the Apes, those scientists think that these bigger evolutionary gaps happen through the insertion of an infectious agent, like a retrovirus. The theory is that every once in a while, a virus can insert in a genome and trigger a significant rearrangement of our underlying genetic code. This switch of code can reveal faculties that have been present within the code but have remained hidden or add new stretches of code that can be used by for our benefit.

So, why is this not far-fetched? CRISPR teaches us why. CRISPR is a new and highly effective scientific technique for altering a genome with a deliberate accuracy. CRISPR is an acronym that stands for the particular specialized regions of DNA separated by spaces in a genome which are the targets of that technique. Scientists have devised a means of inserting carefully tailored clusters of DNA into these areas by taking advantage of those repeating segments and the spaces in between them. Importantly though, those spaces are areas of previously inserted viral code as a result of prior infectious attacks by viruses or retroviruses. Over the course of evolution, new virus attacks have yielded new spacers. Scientists are able to use small segments of genetic code to precisely insert or delete genetic code based on those spaces and the types of code in between. The important point is that the mechanics of CRISPR is very similar to how infectious code has always interacted with our native DNA.

So what does this mean for evolution? Quite directly, if Man can do it, then Nature has always done so. Man is not yet capable of devising a method of adjusting any genetic code that Nature has not already provided. When scientists inserts bits of genetic code to correct a problem, they are mimicking a natural process and making adjustments to it fit our ends.

Certainly, any of the CRISPR alternations may yield substantial benefits. But, the process is still very new with wide-ranging consequences. If genetic syndromes that affect how we look, act, or metabolize can be adjusted by Man by a focused switch of code, then Nature has done it, too. Not often, surely, but just enough to yield the complex organisms that we can observe.

A salient question arises. What parameters and controls ought to be placed on this technique that so powerfully mimics the actual mechanisms of evolution? Do we, as yet, understand the entirety of its implications or are we inadvertently exposing ourselves to substantial unintended consequences?

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From COSMOS: “CRISPR gene editing puts the brakes on cancer cells

COSMOS

26 May 2017
Anthea Batsakis

A cancer cell in the process of division. Knocking out the Tudor-SN protein might have stopped things getting this far. Steve Gschmeissner / Getty

Cancer cells are known for their fast and rapacious growth, but a new technique to slow them down may one day offer new treatment options.

Scientists from the US have discovered a protein called Tudor-SN linked to the “preparatory” phase of cell life – when cells prepare to divide and spread.

Using the gene-editing technology CRISPR, the researchers removed the protein, which is more abundant in cancer cells than healthy cells, and found cancer cell growth was effectively delayed.

The research team, led by Reyad Elbarbary and Keita Myoshi from the University of Rochester, in New York, made its findings in a laboratory using cells from kidney and cervical cancers.

While the technique is still far from human trials, the researchers report in the journal Science that their findings could potentially be used as a treatment option.

See the full article here .

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From AAAS: “First proposed human test of CRISPR passes initial safety review”

AAAS

Jun. 21, 2016
Jocelyn Kaiser

McGovern Institute for Brain Research at MIT

By Jocelyn KaiserJun. 21, 2016 , 5:15 PM

A cancer study that would represent the first use of the red-hot gene-editing tool CRISPR in people passed a key safety review today. The proposed clinical trial, in which researchers would use CRISPR to engineer immune cells to fight cancer, won approval from the Recombinant DNA Advisory Committee (RAC) at the U.S. National Institutes of Health, a panel that has traditionally vetted the safety and ethics of gene therapy trials funded by the U.S. government and others.

Although other forms of gene editing have already been used to treat disease in people, the CRISPR trial would break new ground by modifying three genes at once, which has not been easy to do until now. The study has also grabbed attention because—as first reported by the MIT Technology Review—tech entrepreneur Sean Parker’s new $250 million Parker Institute for Cancer Immunotherapy will fund the trial.

“It’s an important new approach. We’re going to learn a lot from this. And hopefully it form the basis of new types of therapy,” says clinical oncologist Michael Atkins of Georgetown University in Washington, D.C., one of three RAC members who reviewed the protocol.

The proposed CRISPR trial builds off the pioneering efforts of Carl June and others at University of Pennsylvania (UPenn) to genetically modify a cancer patient’s own immune cells, specifically a class known as T cells, to treat leukemia and other cancers. For the CRISPR trial, a UPenn-led team wants to remove T cells from patients and use a harmless virus to give the cells a receptor for NY-ESO-1, a protein that is often present on certain tumors but not on most healthy cells. The modified T cells are then reinfused back into a patient and, if all goes well, attack the person’s NY-ESO-1-displaying tumors. The UPenn team has already tested this strategy in a small clinical trial for multiple myeloma. But although most patients’ tumors initially shrank, the reintroduced T cells eventually became less effective and stopped proliferating.

To boost the staying power of the engineered T cells, the UPenn group wants to use CRISPR to disrupt the gene for a protein called PD-1. The protein sits on the surface of T cells and helps dampen the activity of the cells after an immune response, but tumors have found ways to hide from T cell attack by flipping on the PD-1 switch themselves. (Drugs that block PD-1 eliminate this immune suppression and have proven to be are a promising immunotherapy cancer treatment.)

June’s team also wants to knock out the genes that code for the two proteins that make up a T cell’s primary receptor so that the engineered NS-ESO-1 receptor will be more effective. To do this, they will introduce into the T cells so-called guide RNAs, which tell CRISPR’s DNA-snipping enzyme, Cas9, where to cut the genome.

The 2-year trial will treat 18 people with myeloma, sarcoma, or melanoma, who have stopped responding to existing treatments, at three sites that are members of the Parker Institute—UPenn, the University of San Francisco in California, and the University of Texas MD Anderson Cancer Center in Houston. June pointed out to RAC that his team already has experience with gene editing. They have used a different technique, called zinc finger nucleases, to disrupt a gene on T cells that HIV uses to enter the cells. In a small trial, this strategy appeared to be safe and has shown promise for helping HIV patients. Those data suggest that CRISPR gene editing should be safe in humans, June said.

To confirm that, researchers conducting the CRISPR trial will look for signs of an immune reaction to the Cas9 enzyme, which comes from a bacterium. They will also look for evidence that it has made cuts in wrong place, potentially creating or triggering a cancer gene. When the UPenn team recently used CRISPR to edit T cells from healthy donors as test run, they checked the 148 genes they most feared Cas9 would mistakenly slice and only found one cut in a harmless location. For the CRISPR trial, the team will do various tests to watch for uncontrolled growth of the modified T cells. Because they are editing three genes, one RAC member also noted, the team should watch for large swapped chunks of chromosomes.

Another concern raised by several RAC members is that June, who would not treat the cancer patients but would serve as the trial’s scientific adviser, and UPenn have a financial interest in the trial. (June has patents on using engineered T cells to treat cancer and has advised companies developing these treatments.) Some on the panel suggested they were particularly sensitive about such concerns given that it was at UPenn in 1999 that a young man, Jessie Gelsinger, died in a gene therapy trial, setting the field back for years. “Penn does have an infamous history in this regard,” says biomedical ethicist and RAC member Lainie Ross of the University of Chicago in Illinois.

However, others on the panel noted that the university could take various steps to mitigate the conflict of interest, for example by recusing June from specific tasks. UPenn itself should decide whether it can directly treat patients or merely supply the modified T cells to other sites for the trial, RAC concluded. Ultimately, RAC members voted unanimously (with one abstention) to approve the trial.

Although RAC endorsement is a big step, the researchers must now seek approval from their own institutions’ ethics boards and the U.S. Food and Drug Administration. Others are likely nipping at their heels. Many thought the Cambridge, Massachusetts–based biotech company Editas Medicine would conduct the first CRISPR clinical trial—it has announced plans to use CRISPR to treat an inherited eye disease in 2017—but RAC has not yet reviewed a proposal from the company.

See the full article here .

The American Association for the Advancement of Science is an international non-profit organization dedicated to advancing science for the benefit of all people.

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From NC State: “What Is CRISPR? And How Can it Be Used to Turn Genes ‘Off’?”

North Carolina State University

June 13, 2016
Matt Shipman

CRISPR systems have been a hot research topic since they were shown to have utility as genetic engineering tools in 2012. And they’re often explained in a way that most folks can understand. But those explanations often overlook key details – like the fact that scientists are still in the process of discovering the fundamental rules of how these systems work.

For example, here’s a simplified explanation: CRISPR-Cas systems protect bacteria from invaders such as viruses. They do this by creating small strands of RNA that match DNA sequences specific to a given invader. When those CRISPR RNAs find a match, they unleash Cas proteins that chop up the invader’s DNA, preventing it from replicating.

But of course it’s more complex than that. For example, there are six different types of CRISPR systems (that we know of). One of the most widely-studied CRISPR systems is CRISPR-Cas9, which is a Type II CRISPR system.

But the most common CRISPR systems in nature are Type I. And new research from NC State is shedding light on some of the fundamental rules that govern Type I CRISPR systems – such as how long that CRISPR RNA can be, and how changing the length of the CRISPR RNA affects the behavior of the system.

To learn more, we talked to Chase Beisel and Michelle Luo, who recently published a paper on the work in Nucleic Acids Research, in collaboration with two groups at Montana State University. Beisel is an assistant professor of chemical and biomolecular engineering at NC State; Luo is a Ph.D. student in Beisel’s lab.

The Abstract: Why are Type I CRISPR systems of particular interest?
Michelle Luo
Michelle Luo

Michelle Luo: As you mentioned, Type I systems are the most common type of CRISPR-Cas systems. They account for over half of known systems. This is of particular interest as we look into co-opting an organism’s own system for other purposes. While CRISPR-Cas9 is undeniably a revolutionary genetic tool, it relies on importing this foreign Cas9 protein into an organism. This is a non-trivial task. However, if you use an organism’s own CRISPR-Cas proteins, as shown in our earlier work, you can avoid the challenges of expressing a non-natural protein. Because Type I systems are so prevalent, they offer a promising route to explore how a natural CRISPR-Cas system can be exploited for other means.

The Abstract: In your recent work, you were evaluating how and whether you could modify the RNA in Type I CRISPR systems. Specifically, you were looking at whether you could modify the length of RNA in Type I CRISPR systems. Why would you want to change the length of the RNA?

Luo: Two years ago, a number of papers were published detailing the crystal structures of Type I protein complexes that bind and help degrade target DNA. These publications hinted at the CRISPR RNA serving as a scaffold to assemble the different proteins in the complex. In other words, the RNA serves as a framework for these proteins to grab onto. Thus, we hypothesized that if we changed the length of the CRISPR RNA, we could change the size and composition of the Type I protein complex, and possibly the complex’s behavior.

The Abstract: How, or why, might expanding the protein complexes used in DNA recognition be useful?
Chase Beisel
Chase Beisel

Chase Beisel: Going into the project, we didn’t know if the longer RNAs would allow the complex to even assemble, let alone function properly. We were surprised to find that the longer RNAs still formed a stable complex that could bind and direct the cutting of DNA. Because this complex is larger and recognizes a longer target sequence, we originally envisioned that the complex could be used for more specific DNA editing or for controlling gene expression.

The Abstract: When I think of CRISPR, I think of a system that either leaves DNA alone or cuts it up. What do you mean when you say that changing the length of the RNA is more effective at gene repression?

Luo: Your summary is on point. Normally, CRISPR-Cas systems survey the DNA landscape, and if they detects a target, they will cut up the DNA with tiny molecular scissors. If the target is not identified, the DNA will be left alone. Our earlier work demonstrated that we can prevent the cutting of the DNA by removing the scissors from the equation. We do this by deleting the cas3 gene from the genomic Type I locus. Now, instead of cutting the DNA, the CRISPR-Cas system simply binds the DNA. If we direct these modified systems to a gene, it will block the expression of that gene. Our most recent work shows that changing the length of the RNA can affect how strongly that silencing occurs. For certain regions, the longer the CRISPR RNA, the stronger the repression.

The Abstract: Does that make the CRISPR system more specific? I.e., does it allow the system to be more targeted in terms of the DNA it “attacks”?

Beisel: We wondered the same thing. We did in fact explore how longer RNAs impact specificity as part of the publication, although the results were mixed. On one hand, more of the RNA was involved in base pairing, where more base pairing would necessarily mean greater specificity. On the other hand, we found the longer RNAs were accommodating to mismatches with the target sequence, suggesting weaker specificity. In the end, more experiments will be needed to explore the question of specificity and how it impacts any downstream uses of Type I systems.

The Abstract: How might that gene repression function be used? Are there any potential applications?

Luo: Absolutely! This is particularly promising for metabolic engineering. If you want to make a microbial factory to produce a valuable product of interest, such as a biofuel, you have to alter the metabolism of an organism. This requires overexpressing genes that lead to production and turning off genes that compete with production. Our system allows researchers to turn off genes in a way that is potent, site-specific, reversible, and multiplexed. Our latest discovery suggests that you can fine-tune the extent of CRISPR-based gene repression simply by altering the length of the CRISPR RNA. That’s what our recent paper in Nucleic Acids Research is about.

The Abstract: What are the future directions for this research?

Beisel: Aside from the applications Michelle mentioned, we’re interested in why nature only uses RNA of a fixed length, given that longer RNAs make perfectly functional complexes. We’re also interested in whether this phenomenon applies across the many different flavors of Type I systems, from those that use far fewer proteins in the complex to those found in organisms living at extreme temperatures.

See the full article here .

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From AAAS: “Researchers rein in slice-happy gene editor, CRISPR”

AAAS

6 January 2016
Kelly Servick

Adapted from H. Nishimasu et al., Cell, 156, 5 (2014); Wikimedia/Creative Commons

Changes to the DNA-cutting enzyme Cas9 make CRISPR more precise.

Keith Joung remembers the first time he took CRISPR for a spin. In late 2012, the pathologist at Massachusetts General Hospital in Boston assembled the components of the new gene-editing technology and fiddled with the DNA of a zebrafish embryo. “It was so easy to do,” he says. “It was just stunning.”

CRISPR—the highly efficient set of molecular scissors recently selected as Science’s Breakthrough of the Year—might be easy to use, but it’s not perfect. Joung and his colleagues soon found that these scissors could get too slice-happy, cutting DNA in unexpected and unwanted locations. In early experiments, the group observed that these off-target effects could occur at some DNA sites with nearly the same frequency as the desired edits. That’s a problem if CRISPR is to form the basis of human therapies, for example, repairing the defective genes that cause muscular dystrophy or hereditary liver disease. Researchers’ primary concern is that cutting into an unwanted gene could cause uncontrolled growth and cancer.

Now, Joung and colleagues have found a way to make CRISPR more precise. In a new study, they modified its cutting enzyme to reduce off-target effects below detectable levels.

“I think that this is a potential breakthrough,” says Jin-Soo Kim, a molecular biologist at Seoul National University who was not involved with the work. But the quest to perfect CRISPR doesn’t have a clear end. “No drugs are free of off-target effects,” he notes. With CRISPR-based therapies still far from human testing, no one knows just how precise is precise enough.

CRISPR relies on a DNA-cutting enzyme called Cas9 attached to a short strand of RNA that guides it to specific point in the genome. When the RNA finds a complementary—or nearly complementary—sequence, Cas9 makes its slice. There are already several approaches to prevent unintended slicing. Shortening the length of the guide RNA makes it more sensitive to mismatched sequences, but it can also create entirely new off-target effects. Some labs have experimented with a version of Cas9 that cuts through a single DNA strand instead of two. That means two Cas9 enzymes bearing two different guide RNAs have to recognize their target sequences to cut both strands—a more demanding matching process. But doubling the number of RNA guides adds bulk, which could make it harder to deliver a CRISPR-based treatment into cells.

In the new work, published online today in Nature, Joung and colleagues took a different approach. They modified the Cas9 enzyme itself to change the way it interacts with DNA. They first altered some of the “residues” on the enzyme’s surface that presumably help the guide RNA pair with its matching DNA strand. One set of modifications created a new variant of Cas9, called Cas9-HF1, that appears to be much more discriminating in its cuts. The researchers made seven different edits guided by seven different RNA strands, each known to produce off-target effects with Cas9. But Cas9-HF1 showed no detectable off-target effects in six of these cases—and just one errant slice in the seventh, they report. Joung adds that the apparent slice could actually be the result of a sequencing error.

The results come on the heels of a similar feat, led by CRISPR pioneer Feng Zhang of Harvard University and the Broad Institute in Cambridge, Massachusetts, published last month in Science. That team modified Cas9 to change how it interacts with a different part of a cell’s DNA. It, too, dramatically improved CRISPR’s specificity. But it’s hard to compare those results directly with the new paper because they used slightly different methods to measure off-target effects.

Joung claims his group’s measurements are roughly 10-fold more sensitive than the one used in the Science paper. Both studies rely on methods that attach molecular tags to all points in the genome where a double-stranded break has occurred, before sequencing the short, flagged segments to count the cuts in various genes. Joung’s team claims to detect edits that occur in at least 0.1% of the genome. Zhang says the method used in his paper has been validated down 0.3%, and it may be even more sensitive.

Does detecting just a couple of faulty cuts in a thousand matter? Absolutely, Joung says. “A lot of therapeutic strategies envision manipulating millions, tens of millions, even hundreds of millions of cells, potentially. So one in 1000 sounds pretty good, but that number can become quite large.” He argues that the field needs tests that root out these potentially harmful effects at frequencies of 0.01% or even lower.

Others are less focused on increasingly sensitive tests. Because CRISPR will never fully be rid of off-target effects, the key question for a given therapy is not strictly how many unwanted cuts it makes, but whether it disrupts any essential genes, says Jiing-Kuan Yee, a molecular biologist at the research center City of Hope in Duarte, California. Each therapeutic application will require its own carefully selected Cas9 molecule—and modifications like those in the two recent papers might be combined.

“Pretty soon, I think everybody’s going to start using these modified Cas9s,” he says. “The [off-target] problem will still be there, but it’s going to be much, much reduced.”

See the full article here .

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