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2018-06-14| Technology

The CRISPR-Cas9 Report Card

by Rajaneesh K. Gopinath
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By Rajaneesh K. Gopinath, Ph.D.

How did the genome-editing tool, regarded as one of the revolutionary breakthroughs of the century, fare thus far and what’s its future?

 

The Discovery

In the early 1990s, Francisco Mojica, a doctoral student at the University of Alicante, discovered a mysterious array of short, palindromic DNA repeats in the archea, Haloferax mediterranei. Little would he have guessed at the time, the impact it would go on to have over modern science in the ensuing years. In fact, he along with Ruud Jansen proposed the now popular acronym, CRISPR that stands for Clustered Regularly Interspaced Short Palindromic Repeats. Although concurrent observations of CRISPR were made independently by Japanese and Dutch researchers, its function was unknown.

Ten years since these initial observations, numerous key studies demonstrated that CRISPR is an intricately evolved adaptive immune system. A collaborative effort between researchers at Danisco and Université Laval resulted in a seminal article that provided empirical evidence of this phenomenon (1). Detailed investigations of underlying mechanisms revealed that archea and some bacteria collect fragments of invading DNA from previous viral infections and integrate it between their palindromic repeats as spacers. This region, known as the CRISPR locus, is inheritable and therefore confers acquired immunity throughout the lineage. These sequences get transcribed as CRISPR RNAs (CrRNAs) which specifically detects and binds to foreign DNA elements through complementary base pairing. Another important cog in this wheel is the Cas9 protein that functions as a nuclease and cleaves the region where CrRNAs are bound to. Further research uncovered a third player, the trans-activating CRISPR RNA (tracrRNA) which performed two critical roles; CrRNA processing and the formation of Cas9 nuclease complex.

 

Scientific Advancement and Immediate Ramifications

Once understood, researchers quickly applied CRISPR for genome-editing in various model organisms. The realization that it could also be implemented in humans, ushered in the most exciting years in the history of genetic engineering. Yet, it also incited the battle for ownership between prominent institutions. While Jennifer Doudna’s lab at University of California, Berkley collaborated with Emmanuelle Charpentier to comprehensively document CRISPR activity in vitro using bacterial DNA (2), a couple of labs from the east coast had tested it in vivo during the same time. Feng Zhang of MIT’s Broad Institute and George Church of Harvard University successfully demonstrated gene editing in mouse and mammalian cells and published their findings as back to back articles in the same issue of Science (3,4).

Another group that concurrently demonstrated the CRISPR system in vitro but is often overlooked by the media is that of Virginijus Siksnys, whose manuscript faced rejections and eventually got published after Doudna’s lab (5). Though many groups shaped the rapidly growing CRISPR field and patented on its distinct aspects by then (6), UC Berkley and Broad Institute disputed over the ultimate rights for gene editing. Despite the fact that Jennifer Doudna filed for the patent first in May 2012, Feng Zhang claimed of demonstrating gene editing in higher eukaryotes and applied for his own patent under an expedited review program in December. He eventually received the rights from the US Patent and Trademark Office (USPTO) in 2014. Subsequently, Berkeley filed for an investigation and this led to a patent interference proceeding. After a series of bitter legal battles, in 2017 the Patent Trial and Appeal Board (PTAB) ruled in favor of Broad. Soon afterward, this decision was further appealed by Berkeley at the US Court of Appeals for the Federal Circuit. The world now awaits the decision of the federal appeals court.

Pioneering researchers in CRISPR technology: (from left) George Church, Jennifer Doudna, Emmanuelle Charpentier, and Feng Zhang (Image Source: https://geneonline.news/index.php/2017/04/10/crispr-patent-war-2016-to-2017/)
Pioneering researchers in CRISPR technology: (from left) George Church, Jennifer Doudna, Feng Zhang, and Emmanuelle Charpentier/Image Source: geneonline.news
 
Achievements and controversies

Meanwhile, the scientific world employed this powerful tool to make remarkable progress, out of which many made the headlines (7). For instance, researchers used CRISPR to cure muscular dystrophy in mice, excise HIV out of human immune cells and completely shut down HIV-1 replication in infected mice cells. They were also used to alter pig genomes in an effort to make their organs suitable for human transplant. Mosquitoes were successfully edited to become poor hosts of the malarial parasite. Additionally, a few applications spurred bioethical debates as well. CRISPR was successfully used to edit human embryos by a group of Chinese scientists sparking a moratorium proposed by Jennifer Doudna.

In due course, many gene editing companies such as Caribou Biosciences, Editas Medicine, CRISPR Therapeutics and Intellia Therapeutics were founded. Despite the initial rise, their value suffered at the stock market due to patent battles. Moreover, recently a few reports threatened to break the CRISPR bubble. A 2017 paper reported that CRISPR–Cas9 caused unexpected off-target changes in mice (8). It was instantly criticized and was duly retracted last month due to “insufficient data to support the claims” (9). In contrast, the discovery of Cas9 immunity in humans was considered rectifiable (10). Using pluripotent stem cells and retinal cells respectively, both studies arrive at similar conclusions that the tumor suppressor p53 reduces the efficiency of CRISPR-Cas9.

In a sudden turn of events, CRISPR, at the moment, faces a much serious obstacle. Two new reports, one each from Novartis and Karolinska Institute is currently rocking the world. Published on Monday, in the June 2018 edition of Nature Medicine (11,12), the news has already hit CRISPR stocks. Using pluripotent stem cells and retinal cells respectively, both studies arrive at similar conclusions that the tumor suppressor p53 reduces the efficiency of CRISPR-Cas9. This is because gene editing causes p53-mediated DNA damage response resulting in either DNA repair or cell death. Therefore, the cells that eventually survive might potentially harbor p53 mutations leading to cancer. This obviously is a cause for concern in administering gene therapy.

 

Future Implications

In conclusion, this technology is well worth its hype. Various people have immensely contributed to the CRISPR craze, culminating in the birth of a dedicated bimonthly called CRISPR journal. It is a relatively simpler yet powerful alternative to traditional gene editing. Besides, numerous innovations to the technology hold promise for medical advances. CRISPR 2.0, which precisely edits a single base, CRISPR-Cas13, an RNA editing technique and the CRISPR/Cas9-APEX or Caspex, a technique that is touted to replace the Chromatin Immunoprecipitation in isolating DNA-associated proteins are all here to stay. Nevertheless, increasing scientific evidence reminds us to proceed with the utmost caution and calls for responsible handling by its growing number of stakeholders.

 

References

1) https://www.ncbi.nlm.nih.gov/pubmed/17379808/
2) https://www.ncbi.nlm.nih.gov/pubmed/22745249
3) https://www.ncbi.nlm.nih.gov/pubmed/23287718
4) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/
5) https://www.ncbi.nlm.nih.gov/pubmed/22949671
6) https://www.liebertpub.com/doi/full/10.1089/crispr.2017.0013
7) https://timelines.issarice.com/wiki/Timeline_of_CRISPR
8) https://www.ncbi.nlm.nih.gov/pubmed/28557981
9) https://www.nature.com/articles/nmeth0518-394a
10) https://www.statnews.com/2018/01/08/immunity-crispr-cas9/
11) https://www.nature.com/articles/s41591-018-0050-6
12) https://www.nature.com/articles/s41591-018-0049-z

 

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