Why CRISPR Gene-Editing Technology Is Captivating Research Field

Tyler Jacks, PhD

CRISPR is a revolutionary new gene-editing technology that has taken the medical research community by storm.

Although researchers have been exploring gene editing for more than 40 years, scientists say the CRISPR technology offers game-changing methods for anticancer research as well as a host of other applications. CRISPR, which is an acronym for clustered regularly interspaced short palindromic repeats, is also referred to as the CRISPR-Cas9 system. It consists of repeating sequences of genetic code, interrupted by spaces or spacer sequences. The technology allows for rapid and precise editing or alteration of a cell or organism’s genome, inactivating or repairing genes as needed by changing the DNA sequences.

The system is “borrowed” from a biological phenomenon in bacteria, whereby bacteria can defend themselves against invading viruses using an enzyme called Cas9 and a piece of RNA called a guide RNA, or gRNA. The gRNA finds the target sequence and binds to it.

The Cas9 enzyme, which has been compared to a pair of extremely sharp scissors, follows the gRNA and makes a cut across the two DNA strands. Once the cut is made, the cell tries to repair itself, but it is at this point that scientists can step in and alter the sequence of the gene that can, in turn, change the function of the encoded protein.

Changing the Landscape

The technology has quickly become the “newest and most widely used gene-editing technique,” according to a National Institutes of Health (NIH) committee that recently examined a number of developments in the broader field.¹ CRISPR “has rapidly led to breakthroughs in the editing of genomes of many organisms, including plants, nematodes, flies, fish, monkeys, and human cells,” the panel said.¹According to researchers, CRISPR and functional genomics have changed the cancer research landscape. Researchers have been working on genome engineering since the 1970s but the techniques were inefficient or too difficult to use. “The reason [CRISPR] is exciting is that we have not had the ability to so precisely and so simply manipulate the genomes of cells, mammalian cells in particular, before this time,” Tyler Jacks, PhD, said in an interview with OncLive. “It’s remarkably powerful and beautiful in its simplicity. It’s just such a simple, straightforward system to use, and that is why it’s so popular— because an eighth-grader could do it.”

Jacks is a Daniel K. Ludwig Scholar and investigtor at the Ludwig Center at MIT, and his laboratory has pioneered the use of CRISPR to construct in vivo models of human cancers. “We can think of older genome engineering technologies as similar to having to rewire your computer each time you want to run a new piece of software, whereas the CRISPR technology is like software for the genome. We can program it easily, using these little bits of RNA,” Jennifer Doudna, PhD, explained in a TED Talk late last year.²

Doudna is a professor of Chemistry and of Molecular and Cell Biology at the Department of Chemistry and Chemical Engineering of the University of California, Berkeley, and also is a pioneer in CRISPR technology.

CRISPR was named as one of the Top 10 breakthrough technologies in 2014 and 2016 by MIT Technology Review, and it won the Science Magazine’s Breakthrough Award in 2015, after being runner up in 2012 and 2013.^3,4

Multiple Manipulations

“It’s swept the field, which is pretty remarkable because the first breakthrough paper was only published in the summer of 2012,” Jacks said. “The last I counted, there had been something like 5000 papers since then, using the technology, which is pretty phenomenal.”The ability to make such precise alterations may have been exciting on its own; however, CRISPR has an even greater advantage: more than one gene can be altered at the same time. Considering that most genetic disorders, including cancer, are caused by more than one damaged gene, the ability for multiple manipulations increases CRISPR’s potential utility.

Tumors require at least 3 to 6 mutations for them to become malignant, starting with a mutation that allows them to activate and sustain continuous proliferation.⁵

Human Trials Proposed

The potential of CRISPR to help researchers target several mutations at once speeds up the possibility of finding treatments for individual cancers.The CRISPR-Cas9 technology is still in the research stages and has not yet reached clinical use, but work is moving ahead at a rapid pace. In June, a proposal to use CRISP technology for the first-in-human clinical trial was approved by an NIH panel.⁶ Plans for the complex therapy call for taking NY-ESO-1, a peptide-based form of adoptive immunotherapy already in clinical trials, and disrupting the activity of the PDCD1 gene that encodes PD-1 as well as the TRAC and TRBC genes, which control T-cell receptor (TCR) alpha and beta, respectively.

The FDA must approve the protocol for the trial to proceed. The research team, led by investigators from the University of Pennsylvania, would seek to enroll 18 patients with multiple myeloma, sarcoma subtypes, or melanoma who have progressive, refractory, or metastatic disease.

CRISPR in Laboratory

Meanwhile, researchers in China already have gained approval to proceed with a clinical trial in which 10 patients with non—small cell lung cancer would receive a therapy in which CRISPR technology is used to knock out the PD-1 gene.⁷Scientists in the Cancer Program at the Broad Institute of MIT and Harvard are using CRISPR in their research to try to understand what genes are essential for cancer cell growth. “You can imagine that if you have a cancer cell line and you use CRISPR to eliminate the function of a gene and, as a result, the cells die, that what you can conclude is that these cancer cells are normally dependent in some way on the function of that gene or that protein,” Brett Tomson, PhD, explained in an interview. “A really great thing about this technology is that you can use it at an enhanced scale to assess thousands of different genes and cancer cell lines to reveal the dependencies of every type of human cancer.”

These types of experiments give researchers large data sets that allow them to compare and contrast across different contexts, define what is critical for cancer cell survival, and try to exploit these “Achilles heels” of cancer to combat the disease in patients.

“Additionally, the Broad’s Cancer Program is using CRISPR with cellular and animal models to create and recapitulate critical gene mutations clinically observed in cancer patients, which will allow a better understanding of how they cause cancer,” said Tomson, who is manager of Broad Institute of MIT and Harvard’s Cancer Outreach Solutions.

Researchers first worked with CRISPR researching single-gene disorders, such as muscular dystrophy and sickle cell anemia. In 2015, researchers from Duke University reported successfully treating muscular dystrophy in an adult mouse model.⁸

Market Potential of CRISPR Technology

Doudna believes that there will be a clinical application of the technology within the next 10 years. “I think that it’s likely that we will see clinical trials and possibly even approved therapies within that time, which is a very exciting thing to think about,” she said in her TED talk.The market potential for CRISPR technology is huge, with substantial predicted growth in the industry. According to one analysis, the global genome editing market is expected to reach more than $3.5 billion by 2019.9

“There are several companies that have been started based on CRISPR technology; I know of at least three and I am sure that there are many more,” Jacks explained. “And, while the companies do have cancer programs, they are not focused on cancer alone in looking at short-term clinical applications. Instead, they are focusing on genetic diseases that could be corrected with CRISPR technology, such as sickle cell disease or cystic fibrosis.

Ethical Questions Abound

“The likelihood is that many, if not all, pharmaceutical companies who are interested in cancer will be using this technology in the future,” Jacks said. “In cancer, you often know what the defective gene is. CRISPR could help you correct that gene defect more efficiently and effectively than existing technologies. There are many other diseases that, in theory, could be corrected by CRISPR, and I think that those companies are focused in these areas.”As with many scientific advances, CRISPR is not without some ethical controversies. “Any technology that involves manipulation of the DNA has certain risks,” Jacks said. “I think it’s too early to say how risky this technology is.”

The safety issues involve the off-targeting of genes, which means that the process could affect genes other than the ones being targeted.

Several researchers are investigating this aspect of CRISPR technology. Currently, use of CRISPR-Cas9 in human cells has shown a high frequency of off-target effects, but fewer in cells from mice and zebrafish.¹⁰ In 2015, Chinese researchers attempted to modify the gene responsible for beta-thalassemia in nonviable embryos.

This raised concern in the scientific community.¹¹ In July 2016, Haeussler et al published a study that evaluated CRIPSR-Cas9 predictions to determine off-target and on-target scoring.12 They established a new website (http://crispor.tefor. net) for researchers that predicts off-targets, and a program that helps design, evaluate, and clone guide sequences for the CRISPR/Cas9 system.

The ethical aspect involves the potential for CRIPSR to repair defective genes in an embryo or in the germline of an individual, or germ-line manipulation. Jacks pointed out that in some situations, it may be very reasonable to consider this approach. For example, in a family with BRCA1 mutation that predisposes to breast and ovarian cancer, why would the technology not be used to fix this gene if it were feasible to do so? “That seems quite reasonable and I think that most people are comfortable with that notion,” he said. “The concern, however, is that once you open the door to the germline genetic alteration, what goes through that door? When do you stop?”

The NIH issued a statement last year, outlining its stance on genomic editing in human embryos: “[The] NIH will not fund any use of gene-editing technologies in human embryos. The concept of altering the human germline in embryos for clinical purposes has been debated over many years from many different perspectives, and has been viewed almost universally as a line that should not be crossed.”

The NIH will continue to support research, but “in a fashion that reflects well-established scientific and ethical principles.”13 Patent Fight Over CRISPR The significance of CRISPR became apparent in other ways as several academic investigators made important discoveries at around the same time. Doudna and Emmanuelle Charpentier, PhD, first reported in 2012 that they had been able to cut isolated DNA strands using CRISPR technology.14 They filed a patent in 2013.

However, Feng Zhang, PhD, of the Broad Institute and the Massachusetts Institute of Technology, filed for a patent in 2013, which was granted in 2014. He was the first to engineer the CRISPR system for use in human cells, demonstrating targeted genome cleavage in human and mouse cells. “There are some well-publicized disputes involving a few universities and others around the world who have filed patents roughly simultaneously,” Jacks said. “I think the courts will have their hands full sorting out who did what when, and who deserves the credit.”

What to Do Now

Ironing out who should be granted patents is vital given the enormous market potential of CRISPR. Some companies have raised money in developing gene therapy using the technology.Current research efforts using CRISPR complement other areas of longtime wide scientific interest, such as understanding how to block the function of genes that are critically involved in many cancer types.

As one example, the RAS family of proteins, which includes the gene KRAS, are mutated in many human cancers, including approximately 35% of lung cancers, 45% of colorectal cancers, and 95% of pancreatic cancers.15

“We’re actually measuring the impact of every gene on growth of KRAS-mutant cancer cell lines,” Tina Yuan, PhD, a senior staff scientist at the Broad Institute said in an interview with with OncologyLive.

“By knocking down every gene in the genome in many different KRAS-mutant cancer cell lines, we can identify which genes will kill KRAS-mutant cancers, and those genes would thus represent promising candidates for drug development,” she said.

In the longer term, if researchers could deliver the CRIPSR components to a cancer cell in a patient—for example, a cancer cell with a KRAS mutation—perhaps the RAS gene could be inactivated by CRISPR.

Combatting Resistance in Cancer Patients

“You could do an in vivo gene deletion as a possible cancer therapy,” Jacks said. “We’re not there yet, but there is the prospect of doing that in the future.”Unfortunately, patients with cancer often develop resistance to certain targeted therapies. This relapse occurs because their cancer cells find a way to evade sensitivity to a treatment, often by acquiring specific key gene mutations.

“There’s a big research effort around the world in understanding that kind of acquired drug resistance. Here at the Broad Cancer program, we are conducting research utilizing CRISPR technology to very precisely determine which genes and mutations are influencing the development of resistance to clinical therapies,” Tomson explained.

The Future of CRISPR

“That, in turn, can allow us to think about ways to stop cancer cells from becoming drug resistant in the first place. If many cancer cells in a given cancer type are becoming resistant to a therapy due to the modification of one gene or pathway, maybe it’s worth thinking about a combination approach to target that resistance pathway right at the onset of treatment so that patients’ long-term responses to therapy could be improved.”Scientists know that this is just the beginning of CRISPR technology. One of the first Cas9-like enzymes has been identified, but many labs are working to isolate other versions of Cas9, which can modify DNA in different ways for different applications. For example, CRISPR-Cpf1, which differs from CRISPR-Cas9, is expected to be even more exact in its ability to alter genes.16 “This conversation could be different in a year or two because as this technology continues to evolve that will completely change the realities of what we can accomplish in the lab using CRISPR. Clearly, CRISPR technology has an exciting future,” Tomson said.

References

1. National Academies of Sciences, Engineering, and Medicine. Gene Drives on the Horizon: Advancing Science, Navigating Uncertainty, and Aligning Research with Public Values. Washington, DC: The National Academies Press. 2016. doi:10.17226/23405.
2. Doudna J. How CRISPR lets us edit our DNA. TEDGlobal>London; September 29, 2015; London, UK. http://goo.gl/rehGSl. Talbot D. Precise gene editing in plants. MIT Technology Review. https://goo.gl/jXsPX3. Published February 23, 2016. Accessed August 3, 2016.
3. And Science’s 2015 breakthrough of the year is…Science. http://goo. gl/QhcFTG. Published December 17, 2015. Accessed August 3, 2016.
4. White MK, Khalili K. CRISPR/Cas9 and cancer targets: future possibilities and present challenges. Oncotarget. 2016;7(11):12305-123017.
5. Shaffer AT. First-in-human CRISPR immunotherapy would target PD-1. OncLive.com. http://goo.gl/jggxAW. Published June 24, 2016. Accessed August 9, 2016.
6. Cyranoski D. Chinese scientists to pioneer first human CRISPR trial. Nature. 535(7613):476—477.
7. Nelson CE, Hakim CH, Ousterout DG, et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 2016;351(6271):403-407.
8. Genome editing/genome engineering market by application (cell line engineering, animal & plant genetic engineering), technology (CRISPR, Antisense, TALEN, Zinc Finger Nuclease), & end user (biotechnology & pharmaceutical, CRO)—global forecast to 2019. MarketsandMarkets. Published May 7, 2015. Accessed August 9, 2016.
9. Rodriguez E. Ethical issues in genome editing using Crispr/Cas9 system. J Clin Res Bioeth. 2016;7:266. doi:10.4172/2155-9627.1000266.
10. Liang P, Xu, Y, Zhang X, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 2015;6(5):363-372.
11. Haeussler M, Schönig K, Eckert H, et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 2016;17(1):148. doi:10.1186/ s13059-016-1012-2.
12. National Institutes of Health. Statement on NIH funding of research using gene-editing technologies in human embryos. https://goo.gl/ yhoc86. Published April 29, 2015. Accessed August 9, 2016.
13. Ledford H. Battle over CRISPR patent heats up. Nature. 2016;529(7586):265.
14. National Cancer Institute. The RAS initiative: the problem. http://goo. gl/OtxzW4. Updated September 24, 2014. Accessed August 3, 2016.
15. In CRISPR genome editing, Cpf1, proved its specificity and produced a mutant mouse. Phys Org. http://goo.gl/x5z1tP. Published June 6, 2016. Accessed August 3, 2016.