Can CRISPR Gene-editing Cut it?

 

The gene-editing technology with the unlikely name offers great potential for genetic engineering.

While not yet a household word, CRISPR, the new gene-editing technology, has taken the science of genetic engineering by storm. More precise than competing technologies while involving a fraction of the time and effort, CRISPR is already having a strong impact on genetic research. Scientists are making bold predictions that CRISPR technologies will lead to advances in sectors as diverse as public health, agriculture, and medicine.

In Taiwan, scientists and researchers are already employing the technology in the search for advanced treatments of intractable diseases such as hepatitis and cancer and in the search for higher yielding crops.

“CRISPR makes it so easy to edit the mammalian genome; it is a quite amazing and significant finding,” says Yang Hung-chih, associate professor of microbiology at National Taiwan University’s College of Medicine, who has used CRISPR in research targeting chronic hepatitis infection. “Whoever gets their name as the discoverer of CRISPR will get the Nobel Prize,” he predicts.

Yet for all of the excitement over CRISPR’s potential, it is worth noting that other gene-editing technologies previously were similarly greeted with overwhelming optimism only to falter. According to the Gartner Hype Cycle, exciting new technologies are often pushed up the Peak of Inflated Expectations before falling into the Trough of Disillusionment. In the mid-2000s, RNA interference (RNAi) technology also showed great promise in treating a variety of viral infections, but soon came to be seen as extremely difficult to employ clinically. CRISPR seems to be ascending the Peak of Inflated Expectations. Will it soon fall into the trough?

Possibly, as mixed results in an array of research fields highlight the challenges that genetic engineering faces in implementing CRISPR technologies outside of the laboratory. Groundbreaking research by a team of scientists at the NTU College of Medicine on the use of CRISPR in treating chronic Hepatitis B, publishing in the journal Molecular Therapy – Nucleic Acids, revealed great potential for the technology but also significant limitations. “CRISPR has great potential but there are still some obstacles that we need to overcome to achieve the goals,” says NTU’s Yang.

What is CRISPR?

CRISPR is an acronym derived from “clustered regularly interspaced short palindromic repeats” and pronounced “crisper.” It describes repeating sequences of nucleotides in prokaryotes (bacteria and archaea – single-cell organisms lacking membrane-bound organelles) first observed as early as the 1980s. Later, it was discovered that when bacteria are faced with viral attack, they will transcribe CRISPR RNA that recognize the DNA of the invading viruses (called phages) and bind to it. Enzymes, particularly Cas9, are recruited to the location where the CRISPR RNA is bound to the viral DNA, and acting as a pair of molecular scissors, cut out the DNA of the invading virus, destroying it and providing some degree of immunity to the bacteria.

In 2012, American biochemist Jennifer Anne Doudna of the University of California at Berkeley and French microbiologist Emmanuelle Charpentier jointly published an article in the journal Nature that described their invention of a simplified form of CRISPR called CRISPR/Cas9. This version employed a “guide” RNA sequence that could target specific 20-letter DNA sequences that could then be cut out by Cas9 enzymes. The CRISPR/Cas9 system allows scientists to disable or remove specific DNA sequences, and because of the way that cells repair broken strands of DNA, the system can be used to replace specific sequences with other sequences, analogous to the Find and Replace system in a Word document.

Subsequently, in January 2013, MIT and Harvard professor Feng Zhang published a paper in the journal Science that demonstrated an optimized CRISPR/Cas9 system for mammalian cells and a design for expression in those cells. He was the first to apply for permission to use CRISPR/Cas9 techniques on human cells. (Doudna and Zhang are currently squaring off in the U.S. Patent and Trademark Office over rights to the technology).

Publication of these papers launched the CRISPR revolution, and use of the technology in research has skyrocketed. CRISPR/Cas9 operates at the molecular rather than the cellular level, thereby offering far greater ease of use and accuracy than even formerly leading-edge technologies “zinc-finger nuclease” and TALEN (Transcription activator-like effector nuclease). In the three short years since it was invented, a host of companies have sprung up offering guide RNA (gRNA) molecule sequences online for free or at very low cost. Millions of US dollars have flowed into both research institutes and startups poised to cash in on the technology as it matures. Meanwhile, CRISPR experimentation on human embryos has raised warnings of designer humans.

CRISPR and Hepatitis B

The experience of Taiwanese researchers highlights both the opportunities and the challenges involved with CRISPR technology.

NTU professor Yang recalls that publication of the Feng Zhang paper describing the use of CRISPR/Cas9 in mammalian cells prompted researchers at NTU to investigate its utility in combating chronic hepatitis B infection (CHB). Hepatitis B is a highly destructive viral infection that was once a scourge in Taiwan and remains a huge problem in much of East Asia. Although immunization has reduced the prevalence of the disease, thousands continue to live with it. Yang, who holds a Ph.D. from Johns Hopkins focusing on immunology, has dedicated his career to curing the disease.

Hepatitis B remains an elusive quarry primarily because it can lurk hidden in liver cells (hepatocytes) even while a patient remains healthy, and signs of the virus might not even be evident from blood tests. Further, the virus exists as “covalently closed circular DNA” (cccDNA), a specialized ring form of DNA structure that can occur during propagation of the virus in a cell’s nucleus. “Hepatocytes live very long and divide very slowly,” explains Yang.  “As long as the hepatocytes don’t die, the disease can persist forever.”

Antigens and nucleotide analogues (NA) can prevent the disease from replicating but cannot completely eradicate the virus from the body. The Hepatitis B virus (HBV) therefore almost always rebounds once treatment is discontinued. Thousands of Taiwanese continue to suffer from the disease, and liver cancer and liver cirrhosis – two consequences of HBV infection – remain among Taiwan’s top killers.

“The HBV cccDNA exists in the nuclei of infected hepatocytes, which is a major barrier for treatment,” says Yang. “If we can design a strategy to specifically destroy that DNA, we should be able to cure chronic HBV.”

In early 2013, Yang’s research team began work to test whether CRISPR/Cas9 can destroy the cccDNA of HBV and eradicate the disease from the body. Initially, the team didn’t have a specific RNA sequence to act as its guide RNA, and turned to an online depository for possible candidates. The team then screened the candidates in the laboratory by growing cells in the culture room containing HBV DNA, and testing which HBV gRNA enabled CRISPR to perform best. Of eight candidates, the team narrowed the gRNA sequences down to two for proceeding to experiment on mice.

Research into HBV is hobbled by the fact that the virus naturally infects only two species, humans and chimpanzees, although Southeast Asian tree shrews (tupaia) can also be infected. Mice – called chimera mice – can be bred to contain human liver cells, but this is a costly and time-consuming process.

In the highly competitive arena of genetic engineering, the team wanted to be the first to publish groundbreaking results. Instead of taking the time to breed chimera mice, the team employed a system developed by one of its members, Peter Chen, of using a plasmid to act as a carrier DNA, on which they placed the HBV genome. Yang says they injected the HBV carrier plasmid into the mice so that the livers would become infected and start to express the HBV genome.

The experiment “demonstrated that we can specifically destroy the Hepatitis B virus genome in the liver cells of mice using CRISPR,” Yang notes, but it also highlighted a number of challenges that need to be addressed. The most disturbing are “off-target effects.” Although the accuracy of CRISPR/Cas9 is widely touted, mismatches remain a significant issue. Yang says that in a sequence of 20 nucleotides, the Cas9 will occasionally cut a sequence in which as few as 13 nucleotides match.

“It might target some unexpected part of the human genome,” he explains. “For example, if you cut A region from B region, and then A joins together with C, and this is the totally wrong region – it will cause cancer,” says Yang. “The off-target effects cannot be ignored.”

Scientists are looking into a number of methods for dealing with off-target effects. Ching-yen “Stevie” Tsai, manager of the Transgenic Core Facility (TCF) at Academia Sinica, the lab that produces many of the genetically modified lab mice needed for biomedical research, says that one method for dealing with off-target effects is to decrease the number of nucleotides in the sequence from 20 to 17 or 18. “If you decrease the concentration, you can decrease the chance of mismatch,” she says.

Another method is called “nickase,” which involves cutting – or nicking – only one strand of the double-helix DNA strand at a time in the same region. The nickase method employs two Cas9 enzymes and two gRNA sequences cutting in a defined region.

“The advantage is that you need two guide RNA to determine one site, so the specificity will improve,” observes Yang. On the other hand, TCF’s Tsai notes that the efficiency drops using nickase, as the delivery mechanism needs to be larger. “Efficiency decreases but accuracy increases,” says Tsai.

Yang says that recently published research on genetically engineered Cas9 enzymes also holds promise for improving the specificity of the system.

Delivery of the CRISPR/Cas9 system to the target site can also be a problem, as NTU’s research into HBV infection demonstrated. Yang says that simply injecting the system into the body will not effectively deliver the system to the liver, and “even if you directly inject into the liver you won’t expect the plasmid or delivery vehicle to diffuse throughout the entire liver from the injection site.” NTU’s work on developing a more efficient delivery system is still in the research phase.

Yang says the next stage of NTU research on the treatment of HBV infection will be supported by the efficient breeding of chimeric mice using CRISPR/Cas9 technologies. Academia Sinica’s TCF is already using CRISPR technology to develop genetically modified lab mice with higher efficiency and output and at lower cost. “Before CRISPR, the time it took to disrupt the gene in knockout (specific genes removed from the genome) mice would take at least one year, but after CRISPR we can get the knockout mice in less than three months.”

NTU has its own facility for generating genetically modified mice, and is already using CRISPR/Cas9 to develop chimeric mice containing human liver cells.

Implications for Taiwan

Considering all the research being done on CRISPR at the world’s premier institutes in Europe, the United States, and Japan, the question arises of what contribution Taiwan can make in the field. In terms of research infrastructure, Yang concedes that Taiwan is at a significant disadvantage, and TCF’s Tsai notes that budgets here continue to be stretched.

According to Yang, however, Taiwan does in fact have a number of advantages, particularly in the research of specific diseases such as chronic HBV infection. For one, the small size of the island and the close proximity of research centers with medical centers enables Taiwan’s researchers’ to have easy access to significantly large cohorts of readily observable subjects.

“In the past, Taiwan was an endemic area for HBV,” Yang explains. “We have already established a very big patient cohort, so we have the clinical advantage.” Taiwan also has a number of experienced professors and researchers with expertise in the area of HBV infection and liver treatment, he observes.

“Although the infrastructure for research might not be the best, we have a chance to see where research can be used for clinical application,” he says. “We don’t want to do experiments just to publish a paper. Our purpose is to push this into clinical practice to achieve the ultimate goal of curing chronic HBV infection.”

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