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Gene Therapy

Genome Editing Writ Large

Rapid adoption of CRISPR/Cas9 technology is changing our ability to explore genomics and treat genetic diseases

by Ann M. Thayer
September 7, 2015 | A version of this story appeared in Volume 93, Issue 35


Credit: Darren Weaver/Andrew Sobey/ACS Productions/C&EN
CORRECTION: This video was updated on 10/14/20 to correct video footage of cuttlefish. The original clip showed a species of squid.

Information pouring in from accurate, low-cost gene-sequencing machines is allowing scientists to pose more and better genomic questions. But in the absence of easy-to-use genome-editing tools to experiment with, answers have been hard to come by.

Starting to bridge this gap is a three-year-old editing approach called CRISPR/Cas9. Katrine Bosley, head of the CRISPR start-up Editas Medicine, says it’s “like a seed crystal hitting a supersaturated solution.” Ideas and answers are crystallizing from the “rare intersection of a technology and a readiness of the scientific community to know what to do with that technology.”

Credit: McGovern Institute for Brain Research at MIT
Watch an animation of the CRISPR/Cas9 genome-editing system as it cuts and repairs DNA in living cells.

CRISPR/Cas9 is not the first genome-editing tool. Other systems have been in use for years and are even being tried as therapies. But CRISPR/Cas9 is easier to construct and robust enough to work well from the start in many cell types and organisms.

One academic scientist, Jacob Corn, sees CRISPR/Cas9 as “the democratization of gene editing,” broadening it beyond experts and those who can afford expensive reagents from specialized suppliers. Instead, “they have an idea, and they try it,” he says. “They don’t have to be dependent on another lab to ask the question they want to ask.”

As C&EN explores in the pages to follow, the result has been an explosion in research and commercial use, despite uncertainty about ownership of the technology. Small firms have moved quickly to exploit the approach but are still grappling with translating it into products. Meanwhile, science is gaining from the creation of new animal models of disease even as the possibility for unchecked use clouds the field.

Cas9 creating a double-stranded break in DNA at a precise genomic position using RNA.
Credit: Ian Slaymaker/Lauren Solomon, courtesy of Broad Institute
Streptococcus pyogenes Cas9 protein molecule (space-filling structure) uses RNA (green and gray helix) to guide nuclease activity for editing DNA (red helix). Cas9 creates a double-stranded break at a precise position on the DNA.



CRISPRs—clustered regularly interspaced short palindromic repeats—are segments of DNA that in 1987 were discovered intermingled in the genetic code of bacteria. Found to correspond to stored remnants of viral DNA, they were later determined to be part of primitive bacterial immune systems.

RNA templates transcribed from the CRISPR regions are closely associated with an enzyme known as CRISPR-associated protein 9, or Cas9. If the RNA template finds a match in a viral invader’s DNA, the enzyme chops up the DNA to destroy it.

In the same way, CRISPR/Cas9 can be paired with locations in any genome for use as an editing tool. In 2012, Jennifer Doudna of the University of California, Berkeley; her collaborator Emmanuelle Charpentier, then at Umeå University in Sweden; and coworkers showed they could use a guide RNA (gRNA) sequence to direct Cas9 to targeted sites within prokaryotes, single-cell organisms without a nucleus, and precisely cut the cell’s DNA.

Credit: Adapted from OriGene Technologies
Clustered regularly interspaced short palindromic repeats (CRISPRs) technology employs a guide RNA to direct the Cas9 enzyme (light blue) to a target DNA sequence. Once there, Cas9 will bind when it finds a protospacer-adjacent motif sequence (red) in the DNA and cut both strands, priming the gene sequence for editing.
Schematic showing how CRISPR/Cas9 genome editing works.
Credit: Adapted from OriGene Technologies
Clustered regularly interspaced short palindromic repeats (CRISPRs) technology employs a guide RNA to direct the Cas9 enzyme (light blue) to a target DNA sequence. Once there, Cas9 will bind when it finds a protospacer-adjacent motif sequence (red) in the DNA and cut both strands, priming the gene sequence for editing.

In 2013, Feng Zhang of the Broad Institute of MIT & Harvard, George Church of Harvard University, and Doudna’s group separately showed that the system could edit DNA in eukaryotes, or animal cells, including human ones. It was an early step toward the goal of using the technology to modify genes to treat genetic causes of disease.

By 2014, groups led by Zhang and Doudna had deciphered the structure and inner workings of Cas9. Researchers also have been creating and optimizing gRNAs and tinkering with Cas9 enzymes from different bacterial sources to find the most efficient and precise combinations.

Until CRISPR/Cas9, genome-editing tools were beyond the reach of most researchers. Two existing approaches, zinc finger nucleases (ZFNs) and transcription-activator-like effector nucleases (TALENs), require designing site-specific binding and cutting proteins for every gene target. In contrast, CRISPR/Cas9 uses the same cutting protein regardless of the target. And targeting any gene sequence simply requires synthesizing a matching gRNA of about 20 bases to direct Cas9 to the site.

With this capability, a researcher can use a single gRNA to make one cut to deactivate gene function. With two precise cuts using two gRNAs, along with natural cellular repair mechanisms, researchers can remove, repair, or insert genetic information. Multiple gRNAs open the door to simultaneously changing multiple genes.

Scientists quickly began using the technology to more easily decipher the function of all parts of the genome and to explore disease states. They have used CRISPR/Cas9 to edit genes in insects, plants, fish, rodents, and monkeys. Severe combined immunodeficiency disease, sickle-cell anemia, and cystic fibrosis are already being targeted.

At the Broad Institute, a program called the Genetic Perturbation Platform engineers and uses CRISPR/Cas9 to probe the biology of cells. UC Berkeley and UC San Francisco launched the Innovative Genomics Initiative (IGI) in 2014 to promote genome-editing technology across academic and commercial communities. And the individual labs of Doudna, Zhang, and Charpentier, who is now at Germany’s Helmholtz Centre for Infection Research, are running neck and neck to further develop the technology.



In 2012, Doudna, Charpentier, and coworkers filed a provisional U.S. patent application on the fundamental CRISPR/Cas9 technology. Zhang filed his own seven months later. Throughout 2013, and after publication of the work in human cells, everybody amended their applications.


Highlights in the short history of CRISPR/Cas9

◾ Clustered regularly interspaced short palindromic repeats (CRISPRs) are described (J. Bacteriol., 169, 5429)
◾ CRISPRs are recognized as present throughout prokaryotes (Mol. Microbiol., DOI: 10.1046/j.1365-2958.2000.01838.x)
◾ The name CRISPR is coined; CRISPR-associated (Cas) genes are defined (Mol. Microbiol., DOI: 10.1046/j.1365-2958.2002.02839.x)
◾ CRISPRs are found to contain viral sequences; adaptive immune function is proposed (Multiple references)
◾ CRISPRs are found to act in bacterial immune systems (Science, DOI: 10.1126/science.1138140)
◾ CRISPR/Cas cleaves target DNA (Nature, DOI: 10.1038/nature09523)
◾ Guide RNA processing is deciphered (Nature, DOI: 10.1038/nature09886)
◾ CRISPR systems are modular and can be expressed in other organisms (Nucleic Acids Res., DOI: 10.1093/nar/gkr606)
◾ Caribou Biosciences is founded
◾ Cas9 is characterized as an RNA-guided DNA endonuclease (Science, DOI: 10.1126/science.1225829)
◾ Doudna and Charpentier, Zhang file for patents
◾ Site-specific Cas9 genome engineering is done in eukaryotic cells (Science, DOI: 10.1126/science.1231143 and 10.1126/science.1232033; eLife, DOI: 10.7554/elife.00471)
◾ CRISPR Therapeutics is founded
◾ Editas Medicine is founded
◾ Genetic screen in human cells is done using CRISPR/Cas9 (Science, DOI: 10.1126/science.1246981 and 10.1126/science.1247005)
◾ Crystal structures of Cas9 endonucleases are resolved (Science, DOI: 10.1126/science.1247997)
◾ Crystal structure of Cas9 in complex with guide RNA and target DNA is found (Cell, DOI: 10.1016/j.cell.2014.02.001; Nature, DOI: 10.1038/nature13579)
◾ Intellia Therapeutics is founded
◾ Broad Institute/Zhang receive first patent
◾ University of California Regents request patent-interference proceeding

After requesting a prioritized review, the Broad Institute and Zhang received the first CRISPR/Cas9 patent in April 2014. A year later, the University of California requested that the patent office conduct an interference review of competing claims to determine who invented the technology. Although U.S. patent law has moved to a “first to file” system in which precedence is set simply by the filing date, the competing applications were submitted when a “first to invent” rule still applied.

“If they do declare an interference, this is going to be a really humongous showdown and arguably one of the biggest patent fights in the biotech world,” says Jacob S. Sherkow of New York Law School, where he is an associate professor within its Innovation Center for Law & Technology. The process also could be “the last great interference proceeding for any area of technology.”

Some scientists wonder why the patent office issued Zhang’s patent when his application was filed later. Doudna’s original application focused on using CRISPR/Cas9 in prokaryotes, whereas Zhang’s claimed a method for eukaryotes, especially human cells, Sherkow explains. As a result, the filings may have looked dissimilar to an examiner. Overlapping claims may have come later through “a lot of the nuance in the way the claims were drafted and the way that Doudna’s application had been amended,” he adds.

Both sides have been filing materials to support their claims. Zhang’s proof of inventorship relies in part on his lab notebooks, while Doudna offers comment from University of Utah gene-editing expert Dana Carroll that Doudna’s initial publications enabled others to work in eukaryotic cells. But so many previous scientific references have been cited in the case that they may “end up sinking her application,” Sherkow says.

Although the outcome could be winner takes all, it also could be that no one wins. If Zhang’s patent prevails in an interference trial, Doudna is unlikely to get hers, Sherkow explains. If the win goes to UC, Zhang’s patent would be invalidated, but Doudna’s application would still have to be reviewed, with no guarantee of a patent being awarded.

As a foundational patent, the Broad’s is broad. In the past, far-reaching patents that suffered from vagueness have been invalidated because they didn’t meet the tenet of instructing others how to use the technology. “Enablement is not at issue in this case,” Sherkow says. “Best proof of all is the maybe thousands of labs around the world that use CRISPR every single day.”

Although the technology is considered easy, sorting things out legally will be anything but. If the patent office declares an interference, it could take at least a year and millions of dollars in legal fees to resolve. And although many other patents, particularly on uses of the technology, will be issued—the number of filings has jumped to a few hundred in each of the past two years—many believe a foundational patent could be worth billions of dollars.



The number of publications on CRISPR has risen steeply since 2012.a Through the first half of 2015. SOURCE: Chemical Abstracts Service
Bar graph showing the number of CRISPR publications over that past 10 years.
The number of publications on CRISPR has risen steeply since 2012.a Through the first half of 2015. SOURCE: Chemical Abstracts Service

Despite the intellectual property (IP) complications, the inventors quickly joined with investors to start competing companies. Four leading firms tell C&EN they are confident of their access to foundational technology. In addition, they’ve licensed IP from other universities and companies.

The firms say that IP disputes are common and that this one hasn’t hindered their ability to interest investors or partners. “Many people recognize that this is not a new story in biotech,” says Rachel E. Haurwitz, chief executive officer of Berkeley-based Caribou Biosciences, which she set up with Doudna and two other colleagues in 2011. Eventually, she expects, “these disputes will be resolved in such a way that further enables product development.”

For now, companies are offering few specifics about their programs and partnerships. Caribou has named agriculture, industrial biosciences, therapeutics, and basic research as target areas. In therapeutics, it works with Cambridge, Mass.-based Intellia Therapeutics, which it spun off in 2014.

To translate CRISPR/Cas9 into applications, Caribou’s R&D team has been “identifying critical technical milestones,” Haurwitz says. Although the technology works “astonishingly well, right out of the box,” she says, “a lot of work has to be done to go from early-stage proof of concept to robust industrializable, high-throughput use.”

Caribou has been working on the gRNA to enhance DNA-cutting efficiency and reduce off-target events. It also has developed a cell-engineering process that delivers the Cas9 protein precomplexed with gRNA directly into cells. “In cancer cell lines, as well as human primary cells, this results in the highest efficiencies, as well as the fastest kinetics, of editing,” Haurwitz says.

Although editing rates can be high, the edits may not always alter gene function, she explains. To address this, Caribou is creating analytical methods to identify the outcome of an edit. It then feeds the data into an algorithm to better predict the right gRNAs. It also is developing experimental methods for identifying sites of off-target cuts.

In therapeutics, it’s not yet known what constitutes a “dose” of CRISPR/Cas9 or a “functional cure,” in which a gene is edited in enough cells to eliminate symptoms. Ideally, with all the needed pieces packaged into one delivery vehicle, “we’d treat once, and the patient is therapeutically cured,” Intellia CEO Nessan Bermingham says. If a patient needs repeated doses, the delivery method must be nonimmunogenic.

But how long a therapeutic effect lasts and whether it is in fact a cure depends on the type of cell treated. If the cells are stem cells, or another type of dividing cell, the edit will persist. But if the cell is a somatic, or fully differentiated, cell, the edit will last only as long as that cell survives.

The approach taken will be dictated by the disease, cell type, target, and desired outcome. When possible, manipulating cells ex vivo, or outside the body, may be more manageable than working in vivo. It also may be easier to assess ex vivo whether the edit, such as a simple-to-achieve knockout of gene function, was successful.

Although work is under way on all fronts, initial therapies are anticipated to be ex vivo and for diseases traced back to a single gene. Attempts to tackle diseases caused by multiple genes or requiring more complex gene repair or replacement, as well as in vivo therapies, are further in the future.

Some industry watchers predict that CRISPR-modified chimeric antigen receptor T (CART) cells might reach the clinic before the end of next year, to be followed by CRISPR-edited stem cells in late 2017 or early 2018. In January, Intellia formed a five-year collaboration with Novartis, which is also an investor, on ex vivo applications using CART and hematopoietic stem cells.

Similarly, in May, Cambridge-based Editas set up a collaboration with Juno Therapeutics to create anticancer CART cell therapies. Editas was founded in 2013 by a group from Massachusetts Institute of Technology and Harvard along with Doudna, who later cut ties to the firm.

On its own, Editas is tackling a mutation that causes Leber’s congenital amaurosis (LCA), a rare eye disease, CEO Bosley says. The cells affected are photoreceptors in the retina, which can be reached using a viral vector delivery system injected subretinally. Because these particular cells don’t regenerate, the company hopes “to get a complete enough result with one delivery,” she says.


Getting there involved making and testing hundreds of gRNAs, creating a delivery method, designing analytics to check editing efficiency, and establishing specificity. All this was in service of the first program on LCA, Bosley explains, “but they will also be applicable to programs two through 1,000.”

Later on, addressing traditional pharmacology and safety issues should be relatively clear-cut for experienced managers at the start-ups, says Rodger Novak, CEO of CRISPR Therapeutics. Also operating out of Cambridge, the company has rights to Charpentier’s IP, which she retained separately from the UC Berkeley scientists.

In trying to reduce CRISPR/Cas9 to practice, “we are on a very steep learning curve,” Novak says. But he sees it as only temporary. “It is just a lack of experience because these are the very early days,” he says.



Credit: UC Berkeley
Doudna (from top) and Charpentier are disputing Zhang’s patent claims to the fundamental CRISPR/Cas9 technology.
Jennifer Doudna of UC Berkeley.
Credit: UC Berkeley
Doudna (from top) and Charpentier are disputing Zhang’s patent claims to the fundamental CRISPR/Cas9 technology.

While start-ups grapple with developing therapeutics, existing firms have started selling CRISPR/Cas9-related products. For example, England-based Horizon Discovery sells research reagents and uses CRISPR, along with ZFNs and recombinant adeno-associated virus-mediated gene editing, to generate cell lines and research animals.

Finding the appropriate way to deliver the CRISPR/Cas9 system to a cell is not unlike what was done for other gene-editing methods, says Eric Rhodes, Horizon’s chief technology officer. Injecting the isolated gRNA and protein directly is one option. Others include delivering encoding DNA using a plasmid or viral vector or the encoding RNA so that the cell itself generates the gRNA and protein.

Credit: Hallbauer & Fioretti
Emmanuelle Charpentier of the Helmholtz Center for Infection Research.
Credit: Hallbauer & Fioretti

Each approach has pluses and minuses. The DNA form tends to be the longest-lived, while cells tolerate the RNA best, Rhodes says. But the isolated protein is very effective, has a short half-life, and is the least likely to cause off-target events. “Depending on which one of those is most important and how hard it is to deliver each one of those into your particular cell, you can decide which way to go,” he says.

Compared with ZFNs and TALENs, CRISPR/Cas9 “doesn’t really achieve anything that other technologies didn’t do, but it does them more effectively, faster, and cheaper,” Rhodes points out. “It has quickened the pace of a lot of research.”

Whereas creating a knockout cell line might once have taken three to four months and cost $30,000 to $40,000, it now can be accomplished in six weeks for about $1,000 with CRISPR/Cas9. Even more remarkable, it takes a few months rather than a year or more to produce some genetically modified animals, such as mice, even with multiple changes.

Credit: Broad Institute
Feng Zhang of The Broad Institute and MIT.
Credit: Broad Institute

Not only can suppliers offer a wider range of custom products, but the lower cost and ease of use has made the technology “much more accessible to a much broader audience,” Rhodes says. For Horizon, this has opened up the academic market because it has “given academics access to something that before they really just couldn’t afford.”

Academics can also obtain CRISPR/Cas9 reagents, software, and other tools through IGI and Broad researchers. Many of Zhang’s reagents, for example, are available through the nonprofit plasmid repository Addgene.

The nonprofit Jackson Laboratory can provide the “Cas9 mouse,” developed at the Broad and MIT. The mouse is engineered to produce Cas9 in its cells, thereby freeing up space in delivery vectors for more genetic material. For the same space-saving reasons, people have been looking to shave down or find smaller Cas9 proteins.

Some companies have been tentative about entering the reagents market when IP rights are in flux. To ensure that its customers have access, Horizon hedged its bets by licensing CRISPR/Cas9 from Caribou, Harvard, the Broad, and ERS Genomics, which handles Charpentier’s rights.

Others simply licensed the only current patent on the foundational technology. Hudson, N.Y.-based Taconic Biosciences, for example, has a license from the Broad to produce research animals. And Sigma-Aldrich has one so it can sell CRISPR/Cas9 tools and reagents.

As CRISPR/Cas9 tools become more available and the number of publications on the technology increases, many researchers are overwhelmed. Horizon spends time “educating people on what it takes to actually use the technology intelligently so that you get to the right answer in the shortest amount of time,” Rhodes says.

IGI Scientific Director Jacob Corn says many scientists simply don’t know how to begin. To this end, IGI provides resources including workshops and training opportunities through R&D collaborations.

“People who are not normally genome engineers start doing genome engineering,” he says. In helping others develop new CRISPR/Cas9 tools and filling requests for plasmids and protocols, IGI is also seeing interest in engineering animals not typically used as model organisms.

“We want to encourage people to use it, we want to help people get up and running with tools, and we want to teach them how to incorporate it into what they do,” Corn says.



Excitement about CRISPR/Cas9 can lead to high expectations—along with frustration when things don’t go as anticipated. “It’s still a science, and people shouldn’t expect to ‘press the Cas9 button’ and their cells will be edited,” Corn says. “We have done a lot of genome editing, and I am still surprised at how many times we get surprised.”

Some surprises are related to the newness of using CRISPR/Cas9, whereas others come from conducting previously unimagined experiments in new cell types. “While there are a lot of really great opportunities, there is a huge amount that we don’t know,” Corn says. “We are learning very fast, but there are still all these unknown unknowns.”

Adoption and development has moved in leaps and bounds. The excitement stems in part from the fact that “fundamentally, this technology is very robust,” Editas’s Bosley says. “It has worked in many different labs, many different cell types, many different targets, and many different species. And in all these different settings, it is an effective and useful tool.”

Venture capital investors are also excited. In an early funding round, Caribou raised $15 million. Intellia and CRISPR Therapeutics have had two rounds raising $81 million and $89 million, respectively. And Editas just ended a second round, bringing its funding up to more than $160 million.

To date, though, the money has been going to a few companies tied to the inventors. More venture capitalists would like to invest in CRISPR/Cas9 start-ups, the attorney Sherkow says, but they “have decided to sit on the sidelines to see who is going to win the patent fight.” Given that the most money has gone to Editas and that the Broad has widely licensed Zhang’s patent, a loss by Zhang would be a “huge upset to a lot of settled expectations,” he adds.

IP issues could be resolved by the time therapeutics emerge. Biotech firms have moved quickly to determine how they’d like to access the technology, CRISPR Therapeutics’ Novak says. And while larger drug companies were clearly interested in research early on, “it took a while for big pharma to get its head around what you really can do in terms of therapeutic applications,” he adds.

AstraZeneca stands out, announcing four CRISPR/Cas9 research deals earlier this year. Its partners include England’s Wellcome Trust Sanger Institute, the Broad and Whitehead Institutes, IGI, and Thermo Fisher Scientific. Novartis, in addition to working with Intellia, has a research collaboration with Caribou. And Celgene and SR One, GlaxoSmithKline’s venture arm, have invested in CRISPR Therapeutics.

Amid all the excitement, one controversial event looms. In April, Chinese scientists reported that they had attempted to alter a gene associated with a blood disorder in the cells of nonviable human embryos. The move brought to light the ethical implications of editing human genes.

Leading scientists, bioethicists, and legal experts quickly held meetings and penned commentary. Although some seek a moratorium on gene editing of human embryos and reproductive, or germ line, cells, others believe such work should be allowed for research purposes. And Intellia’s Bermingham points out that working in less controversial somatic cells still offers great promise in treating disease.

Bermingham cautions against rushing to judgment and restricting use of CRISPR/Cas9 technology too broadly. “It is critical that we stop and think about where we are and how we want to move forward,” he says. “The technology is only three years old at this point, but it is certainly at a stage of efficacy and application that we really can start to develop therapeutics today.”  


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