What is CRISPR?
Biology has been outshone in recent years. History has been made in physics, with the discovery of the Higgs boson and the detection of gravitational waves. Biological research however, has failed to excite the public as it once did with Dolly the sheep or the Human Genome Project. Although they haven’t been generating headlines, biologists have been making a steady stream of incremental advances. This gradual progress fails to excite the imagination but culminates in something which does. CRISPR, clustered regularly interspaced short palindromic repeats, a technique which enables simple and precise gene editing, is the result of such cumulative work. The power of CRISPR is bringing biology back into the limelight.
CRISPR has become a sensation in biotechnology, labs around the world are embracing its use, companies are scrambling to commercialise it while others warn of ‘designer babies’. The science behind CRISPR however, has humble origins. In 1987, Japanese researcher, Yoshizumi Ishino, noticed some unusual, repeating genetic sequences in E. coli bacteria. He and his team were intrigued,stating that “the biological significance of these sequences is not known” but their investigation went no further.
“Although they haven’t been generating headlines, biologists have been making a steady stream of incremental advances.”
Six years after Ishino’s work, Francisco Mojica, from the University of Alicante, encountered the same sequences in microbes from local salt marshes during his doctoral studies. Mojica found the repeat sequences appearing in the genomes of multiple, distantly related microbes. Mojica characterised these sequences and called them Clustered Regularly Interspaced Short Palindromic Repeats or CRISPR. CRISPR was later shown to be a microbial immune system; bacteria, like us, must fend off viral invaders. Microbes such as bacteria and archaea use CRISPR in nature to seek out and destroy invasive viral DNA or RNA.
This initial identification of CRISPR was conventional biology, describing living systems. In 2012, however, work by Jennifer Doudna and Emmanuelle Charpentier allowed researchers to move from describing, to engineering CRISPR. They introduced a programmable version of CRISPR-Cas9, with which researchers could easily modify genes of their choice. In nature, CRISPR uses two RNA molecules, which bind to DNA, and guide destructive proteins to a target gene. Doudna and Charpentier streamlined this into a system using a single, guide RNA (gRNA). Once the DNA sequence of a target is known, a complementary gRNA is designed and synthesised for use in cells.
This is not the first gene editing techniques, others such as the Zinc Finger Nuclease (ZFN) or TALEN systems preceded CRISPR. ZFNs and TALENs are both man-made, (ZFNs and TALENs) they are constructed by fusing different proteins together. CRISPR, by contrast, is a natural microbial system, it only needs to be tweaked for use in the lab. This makes CRISPR simpler and much cheaper to use than its predecessors. The flexibility of CRISPR allows researchers to engineer or modify genes of their choosing.
CRISPR put to the test
“These germ-line alterations might help to eliminate these genetic diseases from families, such as the blood disorder, beta-thalassemia, targeted by the Sun Yat-sen group.”
Mere months after Doudna and Charpentier’s work was published, it had been trialled successfully elsewhere and other labs began implementing CRISPR-Cas9 in their own research. Excitement around CRISPR spread quickly and the technique gained in popularity. It was soon being tested in multicellular organisms instead of the unicellular microbes in which it originated. In 2015, just three years after CRISPR’s introduction, research by Junjiu Huang and his colleagues from the Sun Yat-sen University in China led to the first use of the technique in human embryonic cells.
Editing of these “germ-line” cells can produce permanent genetic changes which are then inherited by future generations. These germ-line alterations might help to eliminate these genetic diseases from families, such as the blood disorder, beta-thalassemia, targeted by the Sun Yat-sen group. These efforts were however, ultimately unsuccessful. At the end of 2016, a team from Sichuan University, China went on to pioneer its use in human patients at the end of 2016, injecting CRISPR edited cells into a lung cancer patient to ‘switch-off’ the PD-1 gene. Cancer cells exploit PD-1, a gene which inhibits the immune response, in order to protect themselves from the body’s natural defences. CRISPR edited cells prevent cancer from hijacking PD-1. A similar trial has been approved in the United States and is due to start this year.
“This bitter patent battle has attracted much attention, as it could have huge ramifications for the current CRISPR market.”
A market has sprung up around CRISPR, where private companies are seeking to commercialise the technique leading to competition for the lucrative patent rights to the technology. Feng Zhang, from the Broad Institute of MIT and Harvard, an important figure in the initial development of CRISPR-Cas9 has featured in this legal dispute over CRISPR patenting. Doudna and Charpentier are currently involved in an enormous case with Zhang’s group. The groups are competing for the patent rights to CRISPR-Cas9, in anticipation of its medical or industrial applications in the future. Doudna and Charpentier were first to apply for the patent, but the application by Zhang’s group from the Broad went through an accelerated examination process. This expedited application led to the Broad Institute being awarded the patent, a decision to which the Berkeley group responded with a ‘patent interference’ request. The United States Patent and Trademark Office (USPTO) has, since January 2016, been trying to determine who are the creators of the technique and so who should hold the ‘foundational’ patent for CRISPR-Cas9.
This bitter patent battle has attracted much attention, as it could have huge ramifications for the current CRISPR market. Despite the legal rancour, scientists investigating these systems may soon render this corporate clamour for CRISPR-Cas9 irrelevant: Cas9 may be superseded in the near future. It could be just the beginning, the first of many gene editing tools to be plucked from the microbial dark matter. CRISPR-Cas9 is a type of adaptive immune system found in microbes such as bacteria and archaea. It is used naturally by these microbes to remove invasive viral DNA or RNA that is engineered or reprogrammed by researchers to edit genes of their choice. The genetic landscape of the microbial world is, however, astonishingly diverse and Cas9 is by no means the only CRISPR system out there.
“A gradual accumulation of discoveries, each of which appear small on its own, will culminate in a powerful gene editing suite.”
Recent work has seen the CRISPR family expanding, improving the capabilities of the gene editing tools that scientists can use. Research from the Zhang lab discovered CRISPR-Cpf1, which can be used in genomes which are not suitable for Cas9. Cpf1 may also be more efficient at inserting DNA, rather than just removing it. The Zhang lab also discovered the CRISPR-C2c2 variant, which targets RNA instead of DNA. By targeting RNA, C2c2 won’t make permanent edits to the genome. This allows it to make temporary, adjustable changes to a gene, rather than permanently removing it from a cell.
At the end of 2016, Jillian Banfield, a colleague of Doudna’s at UC Berkeley used metagenomics, a technique used to analyse the genomes of microbial ecosystems, instead of individual species. Using these vast datasets is more complex than other methods, but makes a discovery more likely. Metagenomics is useful for uncultivated microbes, organisms which are difficult to grow in culture in the lab. Banfield studied the genomes of exotic microbes, living in toxic water or underground geysers. From these unlikely sources, she uncovered new CRISPR systems, such as CasX and CasY which are minimalistic, more compact than Cas9. It could, therefore, be easier to incorporate them into cells in the lab.
Following the publication of Banfield’s research, work led by Benjamin Rauch from UC San Francisco introduced yet another tool to the CRISPR toolbox, inhibitory ‘off-switch’ proteins. They sifted through the genomes of Listeria bacteria and found ‘AcrIIA’ proteins with which to counteract DNA degradation by CRISPR. Such ‘anti-CRISPR’ proteins are used by bacteria to minimise the risk of destroying their own genes rather than the viruses they are targeting. These new AcrIIA proteins could be used in research to reduce ‘off-target’ effects in gene editing. Unwanted modifications plagued the unsuccessful editing of embryonic cells at Sun Yat-sen University, for example. AcrIIA proteins could help researchers to alter specific genes, without damaging other genes in the process, fine-tuning the control of CRISPR.
These discoveries are interesting but, in isolation, are not quite revolutionary. As more and more CRISPR systems are discovered however, they begin to compose a comprehensive toolbox. A gradual accumulation of discoveries, each of which appears small on its own, will culminate in a powerful gene editing collection. Already, research has yielded CRISPR systems which are big or small, which cut or insert genes, which make temporary or permanent changes, along with an essential off-switch.
These initial searches for CRISPR systems are not exhaustive, there are almost certainly other versions tucked away in the genetic tree of life. We have a small selection now which could expand into a comprehensive, general purpose toolbox in the future.
Biotechnology companies pouncing on the patents for biological processes may seem worrying. Science can however move faster than these organisations. The tree of life has plenty of genetic tricks waiting to be found. With so much discovered in such a short time, there is reason to be optimistic. The initial development of programmable CRISPR was a gradual process that eventually paid off. The platform has been established, the science is just getting started.